Hydrogel materials suitable for lung organoid growth, methods of making and using the same

Abstract

The application provides a hydrogel material suitable for lung organoid growth and a preparation method and application thereof, and through preparation of lung tissue specific decellular extracellular matrix hydrogel, the natural extracellular matrix structure, protein component and solidified growth factor of lung tissue are completely retained, the lung in-vivo microenvironment can be better simulated, cell adhesion, proliferation and correct lineage differentiation can be more effectively induced when the lung organoid is cultured, meanwhile, the hydrogel material has good biocompatibility and adjustable gelation performance, the effect of culturing lung tumor organoid is not obviously different from that of imported Matrigel, the source is more extensive, the cost is lower, the batch stability is higher, and the biological safety risk and component uncertainty brought by animal source tumor extract are avoided, a reliable new platform is provided for standardization culture of lung organoid, drug screening and individualized medical treatment, and the application has potential application value for replacing Matrigel to culture lung organoid.

Description

Technical Field

[0001] This invention relates to the field of biomedical tissue engineering, and in particular to a hydrogel material suitable for the growth of lung organoids, its preparation method, and its application. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide. Due to its high heterogeneity, responses to treatments such as chemotherapy, targeted therapy, and immunotherapy vary significantly among different patients and even among different lesion sites within the same patient. Therefore, developing preclinical models that can accurately predict individualized treatment responses is crucial for achieving precision medicine. Traditional cancer research relies on two-dimensional (2D) cultured immortalized cell lines and human tumor xenograft (PDX) animal models. 2D cell lines undergo significant changes in their genome and phenotype after long-term in vitro culture, failing to represent the complexity of the original tumor. While PDX models retain some tumor heterogeneity, they suffer from high costs, lengthy processing times, inability to fully simulate the human immune system, and low throughput, making them unsuitable for rapid clinical decision-making and large-scale drug screening.

[0003] In recent years, organoids derived from patient tumor tissue have shown great application potential as a novel three-dimensional (3D) culture model. Lung tumor organoids can simulate the histopathological structure, genomic characteristics, and phenotypic heterogeneity of the original tumor in vitro. Currently, the 3D culture substrate widely used in academia and industry is Matrigel, a basement membrane complex extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Although Matrigel provides a favorable biochemical and physical environment for organoid formation due to its rich content of various extracellular matrix (ECM) proteins and growth factors, its inherent batch-to-batch variability, potential biosafety risks from animal-derived pathogens, unclear composition, and high cost have greatly limited the process of lung organoid research moving from the laboratory to standardization and clinical translation. Furthermore, Matrigel is derived from mouse sarcoma, and its ECM components and signaling molecule profiles differ significantly from the natural microenvironment of human lungs. This non-tissue-specific microenvironment may not be able to most effectively induce the proper adhesion, proliferation, and differentiation of lung tissue cells.

[0004] Therefore, there is an urgent need in this field to develop an alternative material with relatively well-defined composition, safety and reliability, controllable cost, and lung tissue-specific biological functions to overcome the above-mentioned shortcomings of Matrigel and provide a better in vitro microenvironment for lung organoid culture and precision treatment research for lung cancer. Summary of the Invention

[0005] The purpose of this invention is to provide a hydrogel material suitable for the growth of lung organoids, its preparation method, and its application, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing a hydrogel material suitable for lung organoid growth, comprising the following steps: (1) The lung tissue was subjected to defatting, decellularization, sterilization and pyrogen removal in sequence, and then freeze-dried to obtain lung decellularized extracellular matrix material; (2) After the lung decellularized extracellular matrix material is crushed, it is digested with acid and pepsin, the supernatant is collected by centrifugation, and the pH is adjusted to 7.0-7.5 to obtain a pregel; (3) The pregel is mixed with a mixed solvent to obtain the hydrogel material; The mixed solvent comprises DMEM culture medium, HEPES, sodium bicarbonate, GlutamAX, and Ham's F-12 nutrient mixture.

[0007] Preferably, the defatting treatment in step (1) includes: placing small pieces of lung tissue in a defatting solution and stirring; the decellularization treatment includes: placing the defatted lung tissue in sodium dodecyl sulfate solution and Triton X-100 solution in sequence and stirring; the sterilization treatment includes: placing the decellularized lung tissue in a chemical sterilization solution containing peracetic acid and ethanol; the pyrogen removal treatment includes: placing the sterilized lung tissue in physiological saline containing sodium hydroxide with a pH of 9.5-10.5.

[0008] Preferably, the concentration of the sodium dodecyl sulfate solution is 0.8%-1.2% (w / v), the concentration of the Triton X-100 solution is 0.8%-1.2% (w / v), and the chemical sterilization solution contains 0.1%-0.4% (w / v) peracetic acid and 6%-10% (v / v) ethanol.

[0009] Preferably, the acid in step (2) is hydrochloric acid with a concentration of 0.008M-0.012M, and the enzyme activity of the pepsin is 2000-2500U / mg; the digestion conditions are stirring at 23℃-27℃ for 16h-32h.

[0010] Preferably, the content of each component in the mixed solvent in step (3) is: 10% (v / v) of 10×DMEM medium, 1.2%-1.5% (w / v) of HEPES, 0.9%-1% (w / v) of sodium bicarbonate, 0.6%-0.65% (w / v) of GlutamAX, and 9%-12% (v / v) of Ham's F-12 nutrient mixture.

[0011] Preferably, after the pregel in step (3) is mixed with the mixed solvent, the mass-volume concentration of the lung decellularized extracellular matrix hydrogel in the hydrogel material is 6-15 mg / mL.

[0012] Preferably, the lung tissue is derived from pigs or humans.

[0013] The present invention also provides a hydrogel material suitable for the growth of lung organoids, which is prepared by a method for preparing hydrogel materials suitable for the growth of lung organoids.

[0014] Preferably, the hydrogel material can form a non-flowing gel at 37°C, and its storage modulus is 80-120 Pa.

[0015] The present invention also provides the application of a hydrogel material suitable for the growth of lung organoids in the preparation of a culture medium for culturing lung organoids.

[0016] The present invention achieves the following beneficial technical effects compared to the prior art: This invention provides a hydrogel material suitable for lung organoid growth, its preparation method, and its applications. By preparing a lung tissue-specific decellularized extracellular matrix hydrogel, this invention completely preserves the natural extracellular matrix structure, protein components, and immobilized growth factors of lung tissue, better mimicking the in vivo microenvironment of the lung. This allows for more effective induction of cell adhesion, proliferation, and correct lineage differentiation during lung organoid culture. Simultaneously, this hydrogel material exhibits good biocompatibility and tunable gelling properties. Its effect on culturing lung tumor organoids is not significantly different from imported Matrigel, but it has a wider range of sources, lower cost, and higher batch-to-batch stability. Furthermore, it avoids the biosafety risks and compositional uncertainties associated with animal-derived tumor extracts. This provides a reliable new platform for standardized lung organoid culture, drug screening, and personalized medicine, and has the potential to replace Matrigel in lung organoid culture. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 HE staining of the decellularized extracellular matrix material of porcine lung prepared in this invention; Figure 2 DAPI staining of the decellularized extracellular matrix material of porcine lung prepared in this invention; Figure 3DNA content detection of the decellularized extracellular matrix material of porcine lung prepared in this invention. Figure 4 This is a verification image of the gelation of the hydrogel prepared in Example 1; Figure 5 The rheological data of the hydrogels prepared in Examples 1 and 2 are shown in the figure. Figure 6 This is a graph showing the in vitro cytotoxicity detection of the hydrogel prepared in Example 1; Figure 7 The image shows the hydrogel prepared in Example 1 used for lung organoid culture. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The purpose of this invention is to provide a hydrogel material suitable for the growth of lung organoids, its preparation method, and its application, so as to solve the problems existing in the prior art.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Example 1 A method for preparing a hydrogel material suitable for lung organoid growth includes the following steps: Step 1: Take fresh pig lungs, rinse the blood off the surface of the tissue with saline solution, and then soak them in 10% saline solution at 4°C for 16 hours. Step 2: After washing three times with PBS solution, the lung mucosa layer is separated from the tissue and then cut into 5mm pieces. 2 The organizational blocks; Step 3: Add isopropanol at a ratio of 1g:5mL, defatt the mixture at 37℃ for 16 hours, and then wash it with purified water 5 times, 30 minutes each time. Step 4: Add 1% sodium dodecyl sulfate solution at a ratio of 1g:10mL and stir at 37℃ for 24h; then replace with 1% Triton X-100 solution at a ratio of 1g:10mL and stir at 37℃ for 24h; then wash with PBS solution for 1 day, changing the solution 8 times during the wash; then wash with purified water for 1 day, changing the solution 8 times during the wash. Step 5: Add chemical sterilization solution at a ratio of 1g:5mL, shake at 4℃ for 2h, then wash 3 times with PBS for 30min each time, and then wash 3 times with purified water for 30min each time; the chemical sterilization solution contains 0.1%-0.4% peracetic acid and 6%-10% ethanol. Step 6: Add sodium hydroxide-containing physiological saline solution at pH=10 at a ratio of 1g:10mL, stir at 37℃ for 4h, then wash with PBS 3 times for 30min each time, and then wash with purified water 3 times for 30min each time. Step 7: Pre-freeze the material obtained in Step 6 at -80℃ for 4 hours, and then freeze-dry it under vacuum at -60℃ for 72 hours; Step 8: After pulverizing the freeze-dried porcine lung decellularized extracellular matrix material, add 0.01M hydrochloric acid containing 1mg / mL pepsin at 10mg / mL, stir and digest at 25℃ for 16h, then centrifuge at 10000rpm / min for 15min, take the supernatant, and adjust the pH to 7.5 with 1M sodium hydroxide solution. Step 9: Add the mixture to make the final gel solution contain: 75% lung decellularized extracellular matrix hydrogel, 10% 10×DMEM medium, 1.35% HEPES, 1% sodium bicarbonate, 0.65% Glutamax, and 12% Ham's 12. Stir at 4°C for 16 hours to obtain a hydrogel material for lung organoid culture.

[0023] Example 2 A method for preparing a hydrogel material suitable for lung organoid growth includes the following steps: Step 1: Take fresh pig lungs, rinse the blood off the surface of the tissue with saline solution, and then soak them in 10% saline solution at 4°C for 16 hours. Step 2: After washing three times with PBS solution, the lung mucosa layer is separated from the tissue and then cut into 5mm pieces. 2 The organizational blocks; Step 3: Add isopropanol at a ratio of 1g:5mL, defatt the mixture at 37℃ for 16 hours, and then wash it with purified water 5 times, 30 minutes each time. Step 4: Add 1% sodium dodecyl sulfate solution at a ratio of 1g:10mL and stir at 37℃ for 24h; then replace with 1% Triton X-100 solution at a ratio of 1g:10mL and stir at 37℃ for 24h; then wash with PBS solution for 1 day, changing the solution 8 times during the wash; then wash with purified water for 1 day, changing the solution 8 times during the wash. Step 5: Add chemical sterilization solution at a ratio of 1g:5mL, shake at 4℃ for 2h, then wash with PBS 3 times for 30min each time, and then wash with purified water 3 times for 30min each time. Step 6: Add sodium hydroxide-containing physiological saline solution at pH=10 at a ratio of 1g:10mL, stir at 37℃ for 4h, then wash with PBS 3 times for 30min each time, and then wash with purified water 3 times for 30min each time. Step 7: Pre-freeze the material obtained in Step 6 at -80℃ for 4 hours, and then freeze-dry it under vacuum at -60℃ for 72 hours; Step 8: After pulverizing the freeze-dried porcine lung decellularized extracellular matrix material, add 0.01M hydrochloric acid containing 1mg / mL pepsin at 15mg / mL, stir and digest at 25℃ for 16h, then centrifuge at 10000rpm / min for 15min, take the supernatant, and adjust the pH to 7.5 with 1M sodium hydroxide solution. Step 9: Add the mixture to make the final gel solution contain: 75% lung decellularized extracellular matrix hydrogel, 10% 10×DMEM medium, 1.35% HEPES, 1% sodium bicarbonate, 0.65% Glutamax, and 12% Ham's 12. Stir at 4°C for 16 hours to obtain a hydrogel material for lung organoid culture.

[0024] Testing and Experiment Experiment 1: HE staining of lung tissue The decellularized extracellular matrix material of porcine lung prepared in Example 1 was placed in 4% paraformaldehyde for 24 hours, and then soaked in purified water overnight. The fixed tissue was dehydrated sequentially with a gradient of alcohols (50%, 75%, 85%, 95%, 100%, 100%) for 5 minutes each. After dehydration, the tissue was transferred to xylene for clearing for 30 minutes. The cleared tissue was then immersed in paraffin I at 60°C for 2 hours, followed by immersion in paraffin II for 2 hours, and then embedded in paraffin and allowed to cool and solidify into a wax block. The wax block was cut into 5μm thick slices using a rotary microtome, attached to glass slides, and baked at 60°C for 60 minutes. The slices were dewaxed with xylene and hydrated with a gradient of ethanols (100%, 95%, 80%, 70%) for 5 minutes each. Hematoxylin staining was added for 5-10 minutes, followed by rinsing with tap water. Differentiation was performed with 1% hydrochloric acid ethanol for 2 seconds, followed by blueing with running water. Immerse in eosin stain for 1 minute to turn the cytoplasm pink. Dehydrate sequentially with graded ethanol, clear with xylene, mount with neutral resin, and observe under a microscope.

[0025] Experimental results: Combined Figure 1 The results showed that the lung tissue decellularized using this method had a clear morphology and no obvious matrix damage.

[0026] Experiment 2: DAPI staining of lung tissue The decellularized extracellular matrix material of porcine lung prepared in Example 1 was placed in 4% paraformaldehyde for 24 hours, and then soaked in purified water overnight. The fixed tissue was dehydrated sequentially with a gradient of alcohols (50%, 75%, 85%, 95%, 100%, 100%), 5 minutes per step. After dehydration, the tissue was transferred to xylene for clearing for 30 minutes. The cleared tissue was then immersed in paraffin I at 60°C for 2 hours, followed by immersion in paraffin II for 2 hours, and then embedded in paraffin and allowed to cool and solidify into a wax block. The wax block was cut into 5 μm thick sections using a rotary microtome, attached to glass slides, and baked at 60°C for 60 minutes. The sections were dewaxed with xylene and hydrated with a gradient of ethanols (100%, 95%, 80%, 70%), 5 minutes per step. Add DAPI staining solution, incubate in the dark for 30 minutes, rinse 2-3 times with PBS to remove excess dye, then mount with water-soluble mounting medium and observe under a fluorescence microscope.

[0027] Experimental results: Combined Figure 2 The results showed that no cell nuclei were observed in the lung tissue material prepared using this decellularization protocol.

[0028] Experiment 3: Detection of residual DNA in lung tissue Experimental method: Accurately weigh 10 mg each of decellularized porcine lung extracellular matrix material and untreated porcine lung. In a 56℃ water bath, digest with proteinase K and RNase to extract genomic DNA (follow the instructions of the DNA extraction kit). Then, the DNA content of the samples was detected according to YY / T1876-2023, "Determination of DNA Residue in Animal-Derived Biological Materials for Tissue Engineering Medical Products".

[0029] Experimental results: Combined Figure 3 The results showed that the DNA content of the lung decellularized extracellular matrix material prepared by this decellularization protocol was much lower than that of the untreated group.

[0030] Experiment 4: Verification of Lung Decellularized Extracellular Matrix Hydrogel Formation Transfer the pre-cooled sol to a 37°C incubator and incubate for 30 minutes to observe whether a non-flowing gel forms.

[0031] Experimental results: Combined Figure 4 The results show that the lung decellularized extracellular matrix hydrogel prepared in this patent can be stably gelled within 30 minutes at 37°C.

[0032] Experiment 5: Rheological Testing Uniform, bubble-free Matrigel and lung matrix gel samples prepared in Example 1 were prepared. A 20 mm diameter parallel plate clamp was selected, and the sample was loaded onto the center of the lower plate. The measurement gap was set, excess sample was scraped off, and the sample was kept at a constant temperature for a period of time to allow it to relax and reach temperature equilibrium. Then, the parameters were set to oscillation mode, 1 Hz, temperature 37 °C, and strain range from 0.1% to 1000% (logarithmic increase), and the gel storage modulus was measured.

[0033] Experimental results: Combined Figure 5 The results showed that the storage modulus of the lung matrix gel prepared in Example 1 was similar to that of Matrigel, both around 100 Pa.

[0034] Experiment 6: In vitro cytotoxicity assay Preparation of extract: The hydrogel material prepared in Example 1 was placed at 37°C for 30 min to form a gel. Then, DMEM culture medium was added at 0.2 g / mL and the mixture was placed at 37°C and shaken at 120 rpm for 72 h. The supernatant was collected by centrifugation.

[0035] Cell preparation: Mouse fibroblasts were revived in DMEM medium containing 10% FBS and cultured at 37°C, 5% CO2, and saturated humidity. After 2-3 passages until the cells reached the logarithmic growth phase, the cells were digested with trypsin, and the cell concentration was collected and adjusted to 1×10⁶ cells / year. 5 The following experiments were conducted using cells / mL.

[0036] Negative sample preparation: DMEM medium containing 10% FBS.

[0037] Positive sample preparation: Prepare DMEM medium containing 10% DMSO.

[0038] Experimental method: 100 μL of cell suspension was added to each well of a 96-well plate, for a total of 1 × 10⁶ cells / well. 4 Cells / well, cultured at 37℃, 5% CO2 for 24 h. After culture, discard the culture medium in the plate, add the extract of each sample, negative sample and positive sample, 6 wells per group, and place in a 37℃, 5% CO2 saturated humidity incubator. The blank sample group only adds DMEM medium containing 10% FBS and does not contain cells. After 24 hours of culture, examine and photograph under a microscope, discard the culture medium in the plate, add DMEM medium containing 10% CCK-8 reagent, and incubate in a 37℃, 5% CO2 saturated humidity incubator for 1 h. After culture, mix by shaking in the dark for 30 min, and measure absorbance at 450 nm using a microplate reader.

[0039] Test results: (e.g.) Figure 6The diagram shows the cell morphology of the product prepared in Example 1 for cytotoxicity testing. Combined with the results in Table 2 (shown below), it is clear that the cell morphology and number in Example 1 (material group) did not change significantly compared to the control group, indicating good biocompatibility.

[0040] Table 2 Cytotoxicity Test Table

[0041] Experiment 7: Lung Tumor Organoid Culture The organoid-containing droplets were mechanically agitated with 1 mL of pre-cooled organoid digestion solution to prevent clumping and aggregation. Digestion was then terminated with pre-cooled digestion termination solution. The supernatant was discarded by centrifugation, and the organoid precipitate was retained. The organoid precipitate was resuspended on ice in the hydrogel prepared in Example 1 (or the matrix gel used as a control), and seeded at a ratio of 1000-2000 cell clusters / 20 μL of hydrogel. The culture plate was then placed in a 37°C incubator for 30 minutes to allow the gel to solidify. After the gel solidified, the well plate was placed in a clean bench, and 500 μL of pre-warmed organoid culture medium was added to the wells. The culture plate was then placed in an incubator for incubation. The culture medium was changed every 2 days during the incubation process.

[0042] Test results: (e.g.) Figure 7 As shown, lung tumor organoids can be successfully cultured in the hydrogel prepared in this patent, and their growth is good.

[0043] This invention has illustrated its principles and implementation methods using specific examples. The descriptions of these embodiments are merely illustrative of the method and its core ideas; furthermore, those skilled in the art will recognize that modifications may be made to the specific implementation methods and application scope based on the principles of this invention. Therefore, the content of this specification should not be construed as limiting the invention.

Claims

1. A method for preparing hydrogel materials suitable for lung organoid growth, characterized in that, Includes the following steps: (1) The lung tissue was subjected to defatting, decellularization, sterilization and pyrogen removal in sequence, and then freeze-dried to obtain lung decellularized extracellular matrix material; (2) After the lung decellularized extracellular matrix material is crushed, it is digested with acid and pepsin, the supernatant is collected by centrifugation, and the pH is adjusted to 7.0-7.5 to obtain pregel; (3) The pregel is mixed with a mixed solvent to obtain the hydrogel material; The mixed solvent comprises DMEM culture medium, HEPES, sodium bicarbonate, GlutamAX, and Ham's F-12 nutrient mixture.

2. The method for preparing a hydrogel material suitable for lung organoid growth according to claim 1, characterized in that, The defatting treatment in step (1) includes: placing small pieces of lung tissue in a defatting solution and stirring; the decellularization treatment includes: placing the defatted lung tissue in sodium dodecyl sulfate solution and Triton X-100 solution in sequence and stirring; the sterilization treatment includes: placing the decellularized lung tissue in a chemical sterilization solution containing peracetic acid and ethanol; the pyrogen removal treatment includes: placing the sterilized lung tissue in physiological saline containing sodium hydroxide with a pH of 9.5-10.

5.

3. The method for preparing a hydrogel material suitable for lung organoid growth according to claim 2, characterized in that, The concentration of the sodium dodecyl sulfate solution is 0.8%-1.2% (w / v), and the concentration of the Triton X-100 solution is 0.8%-1.2% (w / v); the chemical sterilization solution contains 0.1%-0.4% (w / v) peracetic acid and 6%-10% (v / v) ethanol.

4. The method for preparing a hydrogel material suitable for lung organoid growth according to claim 1, characterized in that, The acid mentioned in step (2) is hydrochloric acid with a concentration of 0.008M-0.012M, and the enzyme activity of the pepsin is 2000-2500U / mg; the digestion conditions are stirring at 23℃-27℃ for 16h-32h.

5. The method for preparing a hydrogel material suitable for lung organoid growth according to claim 1, characterized in that, The content of each component in the mixed solvent mentioned in step (3) is as follows: 10% (v / v) of 10×DMEM medium, 1.2%-1.5% (w / v) of HEPES, 0.9%-1% (w / v) of sodium bicarbonate, 0.6%-0.65% (w / v) of GlutamAX, and 9%-12% (v / v) of Ham's F-12 nutrient mixture.

6. The method for preparing a hydrogel material suitable for lung organoid growth according to claim 1, characterized in that, After the pregel described in step (3) is mixed with the mixed solvent, the mass-volume concentration of the lung decellularized extracellular matrix hydrogel in the hydrogel material is 6-15 mg / mL.

7. The method for preparing a hydrogel material suitable for lung organoid growth according to claim 1, characterized in that, The lung tissue was derived from pigs or humans.

8. A hydrogel material suitable for lung organoid growth, characterized in that, It is prepared by the method for preparing hydrogel materials suitable for lung organoid growth as described in any one of claims 1-7.

9. The hydrogel material suitable for lung organoid growth according to claim 8, characterized in that, The hydrogel material can form a non-flowing gel at 37°C, and its storage modulus is 80-120 Pa.

10. The use of the hydrogel material of claim 8 suitable for lung organoid growth in the preparation of a culture medium for culturing lung organoids.

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