Sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold and preparation method thereof

Abstract

This invention discloses a sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold and its preparation method. The method involves first dissolving bacterial cellulose and aminosulfonic acid in N,N-dimethylformamide, heating the solution to obtain a sulfonated bacterial cellulose suspension, then mixing it with a sodium alginate solution and freeze-drying to obtain a three-dimensional scaffold precursor. The precursor is then cross-linked with calcium chloride and freeze-dried again to obtain the sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold. This invention provides a simple preparation method, and the resulting composite three-dimensional aerogel scaffold possesses suitable mechanical properties and porosity, making it suitable for primary hepatocyte growth and effectively promoting the development of primary hepatocytes into organoids.

Description

Technical Field

[0001] This invention relates to a three-dimensional aerogel scaffold composed of sulfonated bacterial cellulose and sodium alginate and its preparation method, belonging to the field of biomimetic scaffolds. Background Technology

[0002] As the largest internal organ, the liver is a key hub for many physiological processes. It not only participates in the metabolism of major nutrients but also plays multiple roles in regulating the immune system and breaking down heterogeneous biological compounds. Current research on the liver utilizes animal models and cell lines; however, animal models suffer from limitations in species variability and the preservation of heritable material, while primary hepatocytes struggle to maintain their metabolic function after in vitro expansion. Organoids are multicellular clusters with similar physiological functions to living organs. With the increasing global burden of liver disease, the demand for liver organoids (3D in vitro liver models) is constantly growing. As a novel tool for hepatotoxicity assessment, liver disease models, and drug screening, organoids are suitable for research on various liver diseases, including metabolic-related fatty liver disease, infectious liver diseases, hereditary liver diseases, and liver cancer.

[0003] Studies have shown that the extracellular microenvironment can regulate organoid quality. The extracellular microenvironment is designed by coordinating the extracellular matrix (ECM) and soluble factors. This requires the use of synthetic and natural ECMs with controlled matrix stiffness and bioactive molecules to mimic normal and diseased liver tissue. Therefore, designing a suitable three-dimensional environment for the differentiation of primary hepatocytes into organoids is a pressing issue that needs to be addressed.

[0004] Bacterial cellulose (BC), produced by bacteria such as *Gluconobacterium acetylene*, exists as an ultrafine network of 3D interconnected nanofibers, characterized by high purity, high porosity, high water retention, high tensile strength, and good biocompatibility. Bacterial cellulose has been extensively studied as a biomaterial in the biomedical field. Sodium alginate (Alg), a natural anionic polysaccharide extracted from various bacteria and brown algae, can interact with divalent cations to form hydrogels. It possesses excellent biodegradability, biocompatibility, non-immunogenicity, and non-toxicity, and is widely used in biomedicine.

[0005] 3D scaffolds composed of bacterial cellulose and sodium alginate exhibit good biocompatibility and flexibility, and are often used in cartilage and bone tissue engineering. However, bacterial cellulose has a simple structure, with its molecular chain containing only a large number of hydrophilic hydroxyl groups, which cannot satisfy the binding between materials, between materials and cells, or between materials and growth factors (Liu, Wei, Haishun Du, Miaomiao Zhang, et al. Bacterial Cellulose-Based Composite Scaffolds for Biomedical Applications: A Review. ACS Sustainable Chemistry & Engineering 8, (2020): 7536–62.). Summary of the Invention

[0006] The purpose of this invention is to provide a three-dimensional aerogel scaffold composed of sulfonated bacterial cellulose and sodium alginate, and its preparation method. The sulfonated bacterial cellulose-sodium alginate composite aerogel scaffold of this invention has a porous three-dimensional structure, possessing both high porosity and low mechanical properties, which is beneficial for the differentiation of primary hepatocytes into organoids.

[0007] The technical solution to achieve the purpose of this invention is as follows:

[0008] A method for preparing a sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold includes the following steps:

[0009] Step 1: Dissolve bacterial cellulose and aminosulfonic acid in N,N-dimethylformamide and react in a sealed container at 50±10℃ for 2-12 hours. Add anhydrous ethanol to terminate the reaction, adjust the pH to 7-8, dialyze, wash, centrifuge and homogenize to obtain sulfonated bacterial cellulose suspension.

[0010] Step 2: Dissolve sodium alginate in phosphate buffer solution to obtain sodium alginate solution;

[0011] Step 3: Mix the sulfonated bacterial cellulose suspension and sodium alginate solution according to the mass ratio of sodium alginate to sulfonated bacterial cellulose of 1-3:2-4, stir evenly, and let stand.

[0012] Step 4: Freeze-dry the uniformly mixed sulfonated bacterial cellulose-sodium alginate solution to obtain a three-dimensional scaffold precursor composed of sulfonated bacterial cellulose-sodium alginate.

[0013] Step 5: Place the three-dimensional scaffold precursor in a calcium chloride solution for crosslinking, and wash it after crosslinking is complete;

[0014] Step 6: Freeze-dry the scaffold obtained in Step 5 again to obtain a sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold.

[0015] Preferably, in step 1, the mass ratio of bacterial cellulose to aminosulfonic acid is 2:7.5.

[0016] Preferably, in step 1, the reaction time is 4 to 8 hours.

[0017] Preferably, in step 1, the concentration of sulfonated bacterial cellulose in the sulfonated bacterial cellulose suspension is 4% (w / v).

[0018] Preferably, in step 1, the dialysis bag used for dialysis has a molecular weight of 8000-14000, and the dialysis time is 5-7 days.

[0019] Preferably, in step 2, the concentration of the sodium alginate solution is 1% to 2% (w / v).

[0020] Preferably, in steps 4 and 6, the freeze-drying temperature is -50℃ to -80℃, and the freeze-drying time is 48 to 72 hours.

[0021] Preferably, in step 5, the concentration of the calcium chloride solution is 1-2 wt%, and the crosslinking time is 1-2 h.

[0022] The present invention also provides a sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold prepared by the above preparation method.

[0023] Furthermore, the present invention provides the application of the above-mentioned sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold in liver organoid culture.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) This invention promotes cell adhesion and proliferation by introducing negatively charged sulfonic acid groups on bacterial cellulose. Sulfonated bacterial cellulose has an ultrafine three-dimensional nanostructure that can induce the proliferation and differentiation of primary hepatocytes. At the same time, sulfonated bacterial cellulose contains a large number of negatively charged sulfonic acid groups, which have a good affinity for cytokines and facilitate the adhesion of growth factors.

[0026] (2) The sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold of the present invention has good biocompatibility and low elastic modulus, which is conducive to the differentiation of primary hepatocytes into organoids. Attached Figure Description

[0027] Figure 1Cross-sectional images of cell viability in a sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold, assessed by Calcein AM / PI staining, are shown. a represents the control group (L-O2 hepatocytes cultured for 2 days in conventional medium); b represents cell morphology and viability of L-O2 hepatocytes cultured for 2 days on an Alg:SBC (1:1) scaffold; c represents cell morphology and viability of L-O2 hepatocytes cultured for 2 days on an Alg:SBC (2:3) scaffold; and d represents cell morphology and viability of L-O2 hepatocytes cultured for 2 days on an Alg:SBC (3:2) scaffold.

[0028] Figure 2 The graph shows the results of hepatocyte proliferation on sodium alginate aerogel scaffolds (Alg) and sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffolds prepared in Examples 2-4.

[0029] Figure 3 The images show the XPS spectra (a) and S2p spectra (b) of SBCs with different degrees of substitution.

[0030] Figure 4 These are field emission scanning electron microscope (FEM) images of the sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold prepared in Example 1 at different magnifications.

[0031] Figure 5 The Fourier transform infrared spectra of sulfonated bacterial cellulose, sodium alginate, and sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffolds are shown.

[0032] Figure 6 The figures (a) and (b) are stress-strain curves and compression modulus diagrams of the sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffolds prepared in Examples 2-4, including SBC, Alg, and examples 2-4.

[0033] Figure 7 This is a morphological image of primary hepatocytes cultured on a three-dimensional aerogel scaffold composed of sulfonated bacterial cellulose and sodium alginate prepared in Example 1, as shown under an optical microscope.

[0034] Figure 8 These are confocal micrographs of primary hepatocytes cultured on a three-dimensional aerogel scaffold composed of sulfonated bacterial cellulose and sodium alginate prepared in Example 2, from left to right: cultured for 1 week, cultured for 2 weeks, and cultured for 3 weeks.

[0035] Figure 9 These are field emission scanning electron microscope (FEM) images of the sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold prepared in Example 3 at different magnifications.

[0036] Figure 10This is a morphological image of primary hepatocytes cultured on a three-dimensional aerogel scaffold composed of sulfonated bacterial cellulose and sodium alginate prepared in Example 3, as shown under an optical microscope.

[0037] Figure 11 These are confocal microscope images of primary hepatocytes cultured on a three-dimensional aerogel scaffold composed of sulfonated bacterial cellulose and sodium alginate prepared in Example 3, from left to right: cultured for 1 week, cultured for 2 weeks, and cultured for 3 weeks.

[0038] Figure 12 These are field emission scanning electron microscope (FEM) images of the sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold prepared in Example 4 at different magnifications.

[0039] Figure 13 This is a morphological image of primary hepatocytes cultured on a three-dimensional aerogel scaffold composed of sulfonated bacterial cellulose and sodium alginate prepared in Example 4, as shown under an optical microscope.

[0040] Figure 14 These are confocal microscope images of primary hepatocytes cultured on a composite scaffold of sulfonated bacterial cellulose and sodium alginate prepared in Example 4, from left to right: cultured for 1 week, cultured for 2 weeks, and cultured for 3 weeks. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings.

[0042] Example 1

[0043] Step 1: Weigh 2g of bacterial cellulose and dry it in a 60℃ oven for 30 minutes.

[0044] Step 2: Dissolve 7.5g of aminosulfonic acid in 150ml of N,N-dimethylformamide.

[0045] Step 3: Transfer the bacterial cellulose to the aminosulfonic acid solution and react in a 250ml sealed volumetric flask at 50℃ for 2h, 12h, 8h and 4h respectively.

[0046] Step 4: Add 50 ml of anhydrous ethanol to the reaction system of step 3 and stir for 10 min.

[0047] Step 5: After the reaction is complete, adjust the pH to 7-8 using NaOH solution.

[0048] Step 6: Transfer the product to a dialysis bag (molecular weight 8000-14000) and dialyze for 5-7 days.

[0049] Step 7: Wash the product from Step 6 three times, then homogenize it to obtain sulfonated bacterial cellulose suspensions with different degrees of substitution.

[0050] XPS was used to compare the types and contents of elements in BC and SBC to determine the degree of sulfation modification. The results are as follows: Figure 3 As shown. Figure 3 In equation a, 285 eV corresponds to the electron binding energy of C, and 531 eV corresponds to the electron binding energy of O. The new peak at 167 eV is attributed to the S2p signal in SBC, indicating that the material contains S atoms in the form of S atoms. 6+ The presence of the form confirmed the presence of the sulfate group, and the four groups of SBCs were named SBC1, SBC2, SBC3, and SBC4 according to the degree of substitution from smallest to largest.

[0051] Table 1 shows the C, O, and S elemental contents of four types of SBCs determined by XPS and the calculated degree of substitution of the sulfate groups. Under fixed conditions of vulcanizing agent content (NH₂SO₃H: glucose monomer = 2:1) and reaction temperature (50℃), four groups of SBC suspensions with different degrees of substitution were prepared by controlling the reaction time. After freeze-drying, four groups of porous sponge-like materials were obtained. The C, O, and S elemental contents on the surface of the four groups of SBCs were determined using XPS, and the degree of substitution of the sulfate groups (DS) was calculated. sul They were named SBC1, SBC2, SBC3, and SBC4 respectively, according to their degree of substitution from smallest to largest.

[0052] Therefore, it can be seen that, when the feed ratio and reaction temperature (50℃) are constant, the effect of increasing the reaction time on the sulfate ester group content is not linear. With increasing reaction time, DS... sul First increase, then decrease. When the reaction time is 4 hours, DS... sul The reaction reached its maximum value in the experimental group. This is likely because with increasing time (2-4 hours), BC and the sulfation reagent mixed more thoroughly, leading to a more complete reaction. However, further extending the reaction time (8-12 hours) resulted in a decrease in DS. sul The O content in SBC also gradually decreased, possibly due to the excessively long reaction time and other side effects, such as dehydration of bacterial cellulose in a highly acidic environment, leading to DS. sul The decrease in O content.

[0053] Table 1 shows the C, O, and S elemental contents of each SBC surface determined by XPS and the degree of substitution calculated using S%.

[0054]

[0055] Example 2

[0056] Step 1: Weigh 2g of bacterial cellulose and dry it in a 60℃ oven for 30 minutes.

[0057] Step 2: Dissolve 7.5g of aminosulfonic acid in 150ml of N,N-dimethylformamide.

[0058] Step 3: Transfer the bacterial cellulose to the aminosulfonic acid solution and react in a 250 ml sealed volumetric flask at 50 °C for 4 h.

[0059] Step 4: Add 50 ml of anhydrous ethanol to the reaction system in step 3 and stir for 10 min.

[0060] Step 5: After the reaction is complete, adjust the pH to 7-8 using NaOH solution.

[0061] Step 6: Transfer the product to a dialysis bag (molecular weight 8000-14000) and dialyze for 5-7 days.

[0062] Step 7: Wash the product from Step 6 three times, then homogenize to obtain a 4% (w / v) sulfonated bacterial cellulose suspension.

[0063] Step 8: Dissolve 2g of sodium alginate powder in 98ml of phosphate buffer solution and stir overnight to obtain a 2% (w / v) sodium alginate solution.

[0064] Step 9: Take 10 ml of sodium alginate solution and 10 ml of sulfonated bacterial cellulose suspension, mix them evenly, and let them stand for 30 minutes, according to the mass ratio of sodium alginate to sulfonated bacterial cellulose of 1:2.

[0065] Step 10: Transfer the mixture of sodium alginate and sulfonated bacterial cellulose to a 96-well plate, 200 μL per well, and let it stand to defoam.

[0066] Step 11: Place the 96-well plate from step 10 into a freeze dryer and freeze dry for 72 hours.

[0067] Step 12: Place the 96-well plate from Step 11 in a 1 wt% calcium chloride solution for crosslinking for 2 hours.

[0068] Step 13: After cross-linking, the scaffold was washed three times with deionized water and then freeze-dried again for 72 hours to obtain a sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold (Alg:SBC(1:1)).

[0069] Field emission scanning electron microscope (FESEM) images of the sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold at different magnifications are shown below. Figure 4 As shown, this indicates that the aerogel scaffold possesses micron-sized pores suitable for cell growth. The Fourier transform infrared spectrum is as follows: Figure 5 As shown, the SBC / Alg composite scaffold contains characteristic peaks of sulfonated bacterial cellulose, with a peak at 1225 cm⁻¹. -1 820cm -1The nearby absorption peaks correspond to the O=S=O asymmetric stretching vibration and the CO stretching vibration. Meanwhile, the SBC / Alg composite scaffold also exhibits the characteristic peak of sodium alginate, at 1601 cm⁻¹. -1 1032cm -1 These are the absorption peaks for amide I and amide III. Figure 6 This diagram illustrates the mechanical properties of the sulfonated bacterial cellulose-sodium alginate composite scaffold. Figure 6 As can be seen in (b), the compressive modulus of the sulfonated bacterial cellulose and sodium alginate composite scaffold is lower than 200 kPa. The lower compressive modulus is conducive to the differentiation of primary hepatocytes into organoids.

[0070] Sulfonated bacterial cellulose-sodium alginate composite scaffolds were used in the culture of primary hepatocytes, with 3 × 10⁶ primary hepatocytes per well. 4 Through an optical microscope Figure 7 and confocal microscope Figure 8 Observation of the growth morphology of primary hepatocytes under different culture cycles revealed that the three-dimensional scaffold showed good support in the differentiation of primary hepatocytes into organoids and can be used as a long-term culture scaffold for liver organoids.

[0071] Example 3

[0072] This embodiment is basically the same as Embodiment 2, except that step 9 is as follows: 8 ml of sodium alginate solution and 12 ml of sulfonated bacterial cellulose suspension are mixed and stirred evenly according to the mass ratio of sodium alginate to sulfonated bacterial cellulose of 1:3, and allowed to stand for 30 minutes. The resulting sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold is named Alg:SBC(2:3).

[0073] Field emission scanning electron microscope images of Alg:SBC(2:3) composite three-dimensional scaffold at different magnifications are shown below. Figure 9 As shown, the Alg:SBC(2:3) composite three-dimensional scaffold also has micron-sized pores suitable for cell growth; Figure 6 As can be seen from (b), the Alg:SBC(2:3) composite stent has the smallest compressive modulus compared to Alg:SBC(1:1) and (Alg:SBC(3:2)), with a compressive modulus of only 20 kPa. Figure 10 and Figure 11 The primary hepatocytes were observed to have the best growth status and significant proliferation on the Alg:SBC (2:3) composite scaffold with low compressibility modulus. As the culture time increased, the primary hepatocytes continued to proliferate and differentiate.

[0074] Example 4

[0075] This embodiment is basically the same as Embodiment 2, except that step 9 is as follows: 12 ml of sodium alginate solution and 8 ml of sulfonated bacterial cellulose suspension are mixed and stirred evenly according to a mass ratio of sodium alginate to sulfonated bacterial cellulose of 3:4, and allowed to stand for 30 minutes. The resulting sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold is named Alg:SBC(3:2).

[0076] Field emission scanning electron microscope images of Alg:SBC(3:2) composite three-dimensional aerogel scaffold at different magnifications are shown below. Figure 12 As shown, the Alg:SBC(3:2) composite three-dimensional scaffold also has micron-sized pores suitable for cell growth; Figure 6 (b) shows that the compressive modulus of the Alg:SBC(3:2) composite scaffold is around 50 kPa. Combined with optical microscopy... Figure 13 and confocal microscope Figure 14 The growth morphology of primary hepatocytes was observed at different culture cycles. The primary hepatocytes showed good growth, and their organoid differentiation morphology could be observed. This indicates that the Alg:SBC (3:2) composite three-dimensional scaffold is suitable for the growth of liver organoids.

[0077] Cell viability was assessed on a sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold to measure its biocompatibility. Cross-sectional observations of cell viability within the scaffold, based on fluorescence detection, were performed. Figure 1 As shown, cell viability in three-dimensional aerogel scaffolds composed of sulfonated bacterial cellulose and sodium alginate with different volume ratios was compared using CCK8 assay. Figure 2 As shown, the results indicate that hepatocytes can grow and proliferate in a sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold from day 1 to day 7.

[0078] Comparative Example 1

[0079] Step 1: Weigh 2g of bacterial cellulose and dry it in a 60℃ oven for 30 minutes.

[0080] Step 2: Dissolve 7.5g of aminosulfonic acid in 150ml of N,N-dimethylformamide.

[0081] Step 3: Transfer the bacterial cellulose to the aminosulfonic acid solution and react in a 250 ml sealed volumetric flask at 50 °C for 4 h.

[0082] Step 4: Add 50 ml of anhydrous ethanol to the reaction system in step 3 and stir for 10 min.

[0083] Step 5: After the reaction is complete, remove the sample and adjust the pH to 7-8 using NaOH solution.

[0084] Step 6: Transfer the product to a dialysis bag (molecular weight 8000-14000) and dialyze for 5-7 days.

[0085] Step 7: Wash the product from Step 6 three times, then homogenize it to obtain a suspension of sulfonated bacterial cellulose.

[0086] Because sulfonated bacterial cellulose suspensions do not possess the properties of hydrogels, they cannot form gels independently.

[0087] Comparative Example 2

[0088] Step 1: Dissolve 2g of sodium alginate powder in 98ml of phosphate buffer solution and stir overnight to obtain sodium alginate solution.

[0089] Step 2: Transfer the sodium alginate solution to a 96-well plate, 200 μL per well.

[0090] Step 3: Add 50 μL of 1 wt% calcium chloride solution to each of the 96 wells and crosslink for 2 h to obtain sodium alginate three-dimensional hydrogel scaffold (Alg).

[0091] The sodium alginate hydrogel prepared in this comparative example is generally hard, has a high Young's modulus, and has too small a pore size, which cannot meet the microenvironment required for the growth of primary hepatocytes.

[0092] Comparative Example 3

[0093] Step 1: Weigh 2g of bacterial cellulose and dry it in a 60℃ oven for 30 minutes.

[0094] Step 2: Dissolve 7.5g of aminosulfonic acid in 150ml of N,N-dimethylformamide.

[0095] Step 3: Transfer the bacterial cellulose to the aminosulfonic acid solution and react in a 250 ml sealed volumetric flask at 50 °C for 4 h.

[0096] Step 4: Add 50 ml of anhydrous ethanol to the reaction system in step 3 and stir for 10 min.

[0097] Step 5: After the reaction is complete, remove the sample and adjust the pH to 7-8 using NaOH solution.

[0098] Step 6: Transfer the product to a dialysis bag (molecular weight 8000-14000) and dialyze for 5-7 days.

[0099] Step 7: Wash the product from Step 6 three times, then homogenize it to obtain a suspension of sulfonated bacterial cellulose.

[0100] Step 8: Dissolve 2g of sodium alginate powder in phosphate buffer solution and stir overnight to obtain sodium alginate solution.

[0101] Step 9: Take 8 ml of sodium alginate and 12 ml of sulfonated bacterial cellulose at a mass ratio of 1:3, mix them evenly, and let stand for 30 minutes.

[0102] Step 10: Transfer the well-mixed sodium alginate and sulfonated bacterial cellulose mixture to a 96-well plate, 200 μL per well.

[0103] Step 11: Add 50 μL of 1 wt% calcium chloride solution to each of the 96 wells and crosslink for 2 h to obtain a sulfonated bacterial cellulose-sodium alginate composite three-dimensional hydrogel scaffold (Alg:SBC(2:3)).

[0104] When Alg:SBC (2:3) hydrogel scaffolds were used to culture primary hepatocytes, cell death began on the second day of culture, and almost all primary hepatocytes died by the fourth day. This may be because the pore size of the Alg:SBC (2:3) hydrogel is too small, hindering the exchange of nutrients between the cells and the environment, thus preventing the primary hepatocytes from absorbing sufficient nutrients and leading to apoptosis.

Claims

1. Use of sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel scaffold in hepatocyte culture, characterized in that, The application discloses a preparation method of a sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel support. Step 1, bacterial cellulose and sulfamic acid are dissolved in N, N-dimethylformamide, and the reaction is carried out in a sealed container at 50±10 DEG C for 2-12 hours; then, the reaction is terminated by adding anhydrous ethanol, the pH value is adjusted to 7-8, and the sulfonated bacterial cellulose suspension is obtained through dialysis, washing, centrifugation and homogenization; Step 2, sodium alginate is dissolved in a phosphate buffer solution to obtain a sodium alginate solution; Step 3, the sulfonated bacterial cellulose suspension and the sodium alginate solution are mixed in a mass ratio of 2:3 of sodium alginate to sulfonated bacterial cellulose, and then stirred uniformly and left to stand; Step 4, the mixed sulfonated bacterial cellulose-sodium alginate solution is freeze-dried to obtain a three-dimensional support precursor of sulfonated bacterial cellulose-sodium alginate composite; Step 5, the three-dimensional support precursor is placed in a calcium chloride solution for cross-linking, and then washed after the cross-linking is completed; Step 6, the support obtained in step 5 is freeze-dried again to obtain the sulfonated bacterial cellulose-sodium alginate composite three-dimensional aerogel support.

2. Use according to claim 1, characterized in that, In step 1, the mass ratio of bacterial cellulose to sulfamic acid is 2:7.

5.

3. Use according to claim 1, characterized in that, In step 1, the reaction time is 4-8 hours.

4. Use according to claim 1, characterized in that, In step 1, the concentration of sulfonated bacterial cellulose in the sulfonated bacterial cellulose suspension is 4% (w / v).

5. Use according to claim 1, characterized in that, In step 1, the dialysis bag with a molecular weight of 8000-14000 is used for dialysis, and the dialysis time is 5-7 days.

6. Use according to claim 1, characterized in that, In step 2, the concentration of the sodium alginate solution is 1%-2% (w / v).

7. Use according to claim 1, characterized in that, In step 4 or 6, the freeze-drying temperature is-50 DEG C to-80 DEG C, and the freeze-drying time is 48-72 hours.

8. The use according to claim 1, characterized in that, In step 5, the concentration of the calcium chloride solution is 1-2 wt%, and the cross-linking time is 1-2 hours.

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