SPF pigskin extracellular matrix hydrogel as well as preparation method and application thereof

Abstract

The invention provides a preparation method of SPF (specific pathogen free) pigskin extracellular matrix (ECM) hydrogel. The preparation method comprises the following steps: S1, collecting SPF pigskin and pretreating; s2, the pretreated SPF pigskin is subjected to degreasing treatment; s3, sterilizing and disinfecting the freeze-dried powder, and then carrying out enzymolysis; s4, adjusting the pH and osmotic pressure of supernate after enzymolysis to obtain a hydrogel product. The source of the hydrogel product does not contain non-biological source materials at all, establishment of a three-dimensional cell culture environment and growth of cultured cells, especially organoids are facilitated, and meanwhile the problems of tumor sources, batch differences, high cost, potential safety hazards and the like of existing hydrogel products such as Matrigel and the like are solved. Experimental results show that the pigskin ECM hydrogel with different concentrations and from different ages can support the growth of intestinal organs, and the number of the organs cultured by the SPF pigskin ECM hydrogel with the concentration of 3 mg / mL is even more than that of Matrigel.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to an SPF porcine skin extracellular matrix (ECM) hydrogel, its preparation method, and its application, particularly an SPF porcine skin ECM hydrogel, its preparation method, and its application in organoid culture. Background Technology

[0002] Organoids are spatial structures formed by inducing cell self-organization and differentiation through ECM and specific growth factors, resulting in organoids containing organ-specific cell types. They possess multiple organ-specific cell types and exhibit similar spatial organization to their corresponding organs; they can reproduce some functions of the corresponding organs, providing a highly physiologically relevant system; compared to traditional two-dimensional cell line models and animal models, organoid models have a composition and behavior closer to physiological cells, a more stable genome, and simpler manipulation, offering significant advantages in terms of cycle time, cost, clinical relevance, and high-throughput screening.

[0003] Since the beginning of research into organoid culture at the start of this century, various organoids derived from normal and tumor tissues have been successfully cultured worldwide, including organoids of the stomach, breast, pancreas, liver, lung, kidney, and skin, as well as corresponding tumor organoids. The combination of organoid culture technology with microfluidics and novel biomaterials in biomedical engineering has led to new concepts such as organoid microarrays, enabling organoid models to more accurately and comprehensively reflect in vivo biological characteristics. Furthermore, the integration of CRISPR / Cas9 genome editing technology with organoid culture systems has opened up new dimensions for precise genetic regulation in a three-dimensional cellular microenvironment, which can be used to establish high-fidelity disease models and provide a highly reliable platform for preclinical validation of gene therapy protocols.

[0004] ECM (Extracellular Memory Gel) is a commonly used natural scaffold material in cell and tissue engineering research, characterized by good mechanical properties, high biocompatibility, and low immunogenicity. Hydrogels prepared using ECM can support organoid culture and are widely used in basic research and product development in animal husbandry, biomedicine, and regenerative medicine. ECM hydrogels possess a certain degree of porosity and rigidity, providing a three-dimensional scaffold for cell growth, mimicking the structural environment of ECM in vivo. Cells can attach to the surface or interior of the hydrogel, interacting with its components to form stable tissue structures, thereby promoting organoid self-assembly and morphogenesis.

[0005] The mechanical properties of hydrogels (such as hardness and viscoelasticity) have a significant impact on the growth and differentiation of organoids. Different types of organoids require hydrogel environments with different hardness. Viscoelasticity also affects organoid development. High viscoelastic hydrogels can promote the growth of liver organoids. The degradability of hydrogels also affects cell migration and proliferation. An appropriate degradation rate can provide space for cell growth and tissue remodeling.

[0006] Naturally derived ECM hydrogels retain the bioactive factors in natural ECM. These factors can interact with receptors on the cell surface, mediating processes such as cell migration, localization, polarization, and signal transduction, and regulating cell growth, differentiation, and functional expression.

[0007] Currently, Matrigel is considered the "gold standard" product for organoid culture, primarily composed of laminin, type IV collagen, and growth factors. However, because Matrigel is extracted from mouse sarcoma, it carries potential risks such as batch-to-batch variability and the spread of animal pathogens. Furthermore, the composition of tumor ECM differs significantly from that of normal tissue ECM, thus failing to provide a tissue-specific microenvironment. Therefore, its tumor origin, batch-to-batch variability, high cost, and safety concerns have consistently drawn criticism from users.

[0008] In addition, the literature also reports the application of ECM hydrogels from other sources in organoid culture.

[0009] Chinese patent application CN119971146A discloses a biomimetic matrix hydrogel for constructing bone organoids and its preparation method. This invention relates to a biomimetic matrix hydrogel for constructing bone organoids and its preparation method. The biomimetic matrix hydrogel is prepared by combining decellularized bone extracellular matrix, salmon DNA network, and calcium phosphate oligomers through a combination of photocrosslinking and self-assembly physical crosslinking. In this invention, the decellularized extracellular matrix in the biomimetic matrix hydrogel can simulate the organic components in natural bone, the calcium phosphate oligomers can simulate the inorganic components in natural bone, and the introduction of the DNA network can provide sufficient phosphate ions for biomineralization. The biomimetic matrix hydrogel can replace Matrigel in bone organoid culture, providing a survival environment similar to bone tissue for three-dimensional cultured cells, enhancing intercellular material exchange and signal transduction, and promoting the proliferation, adhesion, and osteogenic differentiation of bone marrow mesenchymal stem cells, thereby accelerating the biomineralization process. This invention provides a novel material option for the preparation of functionalized bone organoids.

[0010] Chinese patent application CN118359827A discloses a hydrogel for constructing in vitro 3D tumor organoid models, its preparation method, and its applications. The provided OHA CEC hydrogel contains N-carboxyethyl chitosan and oxidized hyaluronic acid, and is a natural OHA CEC hydrogel suitable for in vitro 3D tumor organoid culture. It shows promising application prospects in biomedical fields such as tumor modeling, drug screening, and new drug development.

[0011] Chinese patent document CN114181883B discloses a hydrogel organoid sphere with adjustable hardness containing matrix gel, its preparation method, and its application. The organoid sphere is composed of a hydrogel formed from natural polymer materials such as matrix gel, collagen, and alginate. Cells are uniformly dispersed in the hydrogel solution to form an aqueous phase. Uniform, monodisperse droplets are prepared using microfluidic oil-in-water technology, and then cross-linked with a cross-linking agent to form a uniformly structured cell-carrying interpenetrating network hydrogel organoid sphere. Without changing the matrix gel and / or collagen content, by adjusting the type and concentration of alginate in the aqueous phase and the type of cross-linking agent, hydrogel organoid spheres with accurately adjustable hardness in the range of 100 Pa to 800 kPa can be prepared. This can be used to simulate the hardness of various soft tissue microenvironments in the human body, especially for various tumor organoids, providing experimental evidence for high-throughput drug screening and personalized clinical medication.

[0012] However, the hydrogel components provided in these existing technical solutions all contain non-biological components, such as calcium phosphate oligomers, chitosan, hyaluronic acid, and alginate. In the three-dimensional cell culture process of organoids, the addition of non-biological components will lead to incompatibility of the culture environment and make it impossible to construct the ideal environment for cell growth. Summary of the Invention

[0013] To address the aforementioned technical problems, this invention provides a method for preparing SPF porcine skin ECM hydrogel and its applications. The specific technical solution of this invention is as follows:

[0014] This invention provides a method for preparing SPF porcine skin extracellular matrix (ECM) hydrogel, comprising the following steps:

[0015] S1: Collect SPF pigskin and pre-process it;

[0016] S2: Homogenize the defatted pigskin, freeze-dry the precipitate and grind it into powder;

[0017] S3: Enzymatic hydrolysis of freeze-dried powder after sterilization and disinfection;

[0018] S4: Adjust the pH and osmotic pressure of the supernatant after enzymatic hydrolysis to obtain the hydrogel product.

[0019] Preferably, the enzymatic hydrolysis in step S3 is carried out using 0.05-4 mg / ml pepsin in 0.005-0.1 M hydrochloric acid.

[0020] Preferably, in step S4, pH and osmotic pressure are adjusted using NaOH and PBS to make the hydrogel pH neutral and the osmotic pressure isotonic with that of the cells.

[0021] Preferably, in step S2, Na2CO3 is used as the degreasing solution for degreasing treatment. The solution is continuously stirred at 30-40°C at a stirring speed of 200-800 rpm for 2-6 hours, and the degreasing solution is replaced with an equal amount every hour.

[0022] Preferably, after step S2, the method further includes step S2-1: homogenizing the defatted pigskin, freeze-drying the precipitate and grinding it into powder; the homogenization process includes placing the pigskin in PBS, coarsely beating it with a pulper, homogenizing it in an ice bath at 8000-12000 rpm for 1-5 min, centrifuging the homogenized sample at 4000-10000 rpm for 5-30 min, and discarding the supernatant.

[0023] More preferably, the preparation method of the present invention specifically includes the following steps:

[0024] S1: After collecting SPF adult pig skin, remove the surface hair, fat layer and epidermis, clean it, and cut it into 3.5 mm × 3.5 mm pieces;

[0025] S2: Use 1 / 20 (W / V) 5% Na2CO3 as the degreasing solution, stir continuously at 35℃ and 300 rpm for 3 hours, and replace the degreasing solution with an equal amount every hour; wash the degreased pigskin with pure water for 15 minutes at a time, until the washing solution is neutral, and then use absorbent paper to dry the surface moisture of the pigskin.

[0026] S2-1: Place the defatted pigskin in 1 / 20 (W / V) 1× PBS, coarsely beat it with a homogenizer, then homogenize it at 10000 rpm for 2 min in an ice bath. After homogenization, centrifuge the sample at 6000 rpm for 10 min and discard the supernatant. Freeze-dry the precipitate at -60℃ and below under 1 Pa conditions for 72 h. Grind the freeze-dried sample into powder using a grinder and store it at 4℃ for later use.

[0027] S3: Weigh 400 mg of lyophilized powder sample and sterilize it at room temperature for 2 h with 40 mL of 0.1% peracetic acid + 4% anhydrous ethanol. Wash with the same volume of physiological saline as the sterilizing solution for 15 min / time, 4 times, until the sterilizing solution is completely removed. Wash once with sterile pure water to remove salt. The centrifugation parameters during the washing process are room temperature, 7200 g, 10 min. Prepare 4 mg / mL pepsin with 0.01M HCl. After the enzyme is completely dissolved, filter it through a 0.22 µm filter membrane for sterilization and use it for later use. Enzymatically digest the sterilized and washed lyophilized powder sample with 0.01M HCl and 1 mg / mL pepsin for 48 h. Centrifuge the digested sample at 7200 g for 10 min and collect the supernatant.

[0028] S4: Add 1 / 100 of 1M NaOH and 1 / 9 of 10X PBS to make the hydrogel pH neutral and the osmotic pressure isotonic with that of the cells; after aliquoting the obtained hydrogel sample, store it at -20℃.

[0029] Secondly, the present invention provides an SPF porcine skin ECM hydrogel prepared by the above-described preparation method.

[0030] Thirdly, the present invention provides the application of the SPF porcine skin ECM hydrogel in three-dimensional cell culture.

[0031] Preferably, the application is in organoid culture.

[0032] More preferably, the organoid is an intestinal organoid.

[0033] The present invention has the following beneficial effects:

[0034] The hydrogel product of this invention represents a significant breakthrough in material sourcing, completely eliminating non-biological materials and potentially hazardous biological materials, such as tumor cell extracts and other exogenous polymers. This fundamentally eliminates the risks associated with tumor cell sources and non-biological products like exogenous polymers found in traditional Matrigel hydrogels, which could potentially contaminate cells or hinder cell growth if introduced into a cell culture system. Furthermore, the hydrogel of this invention is derived from SPF (Specific Pathogen Free) pigs, meaning pigs free from specific pathogens, thus ensuring even greater safety.

[0035] The hydrogel product of this invention is extracted solely from pig skin, requiring no other raw materials. This pure material source provides a clean and safe growth environment for cells during cell culture, allowing them to grow and proliferate normally without interference from other components. In organoid culture, the safe hydrogel enables organoids to more realistically simulate the development and function of organs in vivo, providing a solid foundation for disease model construction, drug screening, and regenerative medicine research, and offering reliable assurance for biomedical research and applications.

[0036] The hydrogel product of this invention also demonstrates significant advantages in cost control. Existing hydrogel products such as Matrigel are expensive, mainly due to their complex production processes and scarce raw materials. Matrigel is extracted from mouse tumor cells, and the acquisition process requires not only specialized experimental animal husbandry and management facilities but also limited production volume, resulting in high raw material procurement costs. This high cost puts many research institutions and biopharmaceutical companies under economic pressure when using it, especially for some long-term, large-scale research projects and production activities, where the cost burden is even heavier. Other hydrogel products also require complex extraction procedures and the addition of a large number of auxiliary exogenous non-biological components, which also leads to high costs. However, the applicant has significant advantages in the breeding and raising of SPF pigs. SPF pig skin is a high-quality source of ECM, with great value and application potential as a biomaterial for research and product development. This invention can further broaden its application scenarios, thereby reducing the cost of this invention.

[0037] The hydrogel product of this invention also has advantages in quality batch control. It uses only SPF pigskin as the sole raw material, and the extraction process is a controllable process and control conditions, which can realize large-scale standardized production of the product. Stable quality batches can also ensure the consistency and stability of the product.

[0038] In summary, this invention, using SPF (Special Purpose Fertilizer) pigs as the target, has developed a high-purity, safe, and high-performance SPF porcine skin ECM hydrogel product with promising commercial applications. This product addresses issues such as tumor origin, batch-to-batch variability, high cost, and safety concerns associated with existing hydrogel products like Matrigel. Utilizing SPF experimental pig resources for the development of porcine tissue ECM hydrogels simultaneously promotes the high-value transformation of porcine resources from traditional animal husbandry to the fields of bio-interdisciplinary collaboration and biomedicine.

[0039] Instruction manual with accompanying drawings

[0040] Figure 1 The image shows the final morphology of the SPF porcine skin ECM hydrogel prepared according to the present invention.

[0041] Figure 2This invention demonstrates the DNA residue situation during the SPF pig skin ECM treatment process.

[0042] Figure 3 shows the mechanical properties of the SPF porcine skin ECM hydrogel of the present invention.

[0043] Figure 4 shows a scanning electron microscope image of the SPF porcine skin ECM hydrogel of the present invention.

[0044] Figure 5 shows the frequency of the SPF porcine skin ECM hydrogel of the present invention obtained by scanning electron microscopy and the pore size statistics fitted by Gaussian function modeling.

[0045] Figure 6 The results show that different concentrations of the SPF porcine skin ECM hydrogel of this invention can support organoid growth.

[0046] Figure 7 The photographs show that the SPF porcine skin ECM hydrogel of this invention, from different age sources, can support the growth of intestinal organoids.

[0047] Figure 8 The images show staining of biomarkers that support the growth of intestinal organoids in SPF porcine skin ECM hydrogels of the present invention from different age sources.

[0048] Figure 9 The results of organoid culture using different hydrogels are shown in comparison. Detailed Implementation

[0049] The following detailed description provides further details through specific embodiments. However, it should be noted that the embodiments described below are merely for illustrating the content of the invention and do not represent that the invention is limited to the described embodiments. Therefore, non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention still fall within the protection scope of the invention, and the scope of protection of the appended claims shall prevail.

[0050] Those skilled in the art will understand that the term SPF used in this invention refers to Specific Pathogen Free, which means laboratory animals, plants, or cells that are raised or cultivated under specific conditions and have been explicitly excluded from carrying specific types of pathogenic microorganisms (such as bacteria, viruses, parasites, etc.). The core characteristics of SPF include: 1) Explicit pathogen exclusion: not completely sterile, but proven by testing to not carry pre-defined specific pathogens according to research needs. These pathogens are usually species that can interfere with experimental results, affect the health of organisms, or pose a zoonotic risk; 2) Standardized cultivation environment: They must be raised or cultivated in a strictly controlled barrier environment to prevent contamination by external pathogens and to avoid generating unspecified pathogens themselves; 3) Ensuring experimental reliability: Due to the exclusion of specific pathogen interference, SPF organisms can provide a more stable and consistent experimental model for medical, biological, and other research, ensuring the accuracy and reproducibility of experimental results. The cultivation method and control conditions of SPF pigs used in this invention can be found in the applicant's prior published Chinese patent application CN114711188A, the entire contents of which are incorporated herein by reference.

[0051] The term "extracellular matrix" (ECM) used in this invention refers to the sum of non-cellular components secreted by cells into the extracellular space. It serves as the physical support framework for cells and plays a central role in the regulation of cell function. The ECM is mainly composed of two types of substances: first, fibrous proteins that form the cellular structural framework, including collagen, elastin, fibronectin, and laminin; and second, proteoglycans that act as cell filler materials. Components, including collagen, provide structural support for cell growth, while proteoglycans and other components can absorb water to form hydrogels, providing pressure resistance and water retention, thus contributing to a suitable environment for cell growth.

[0052] The basic process and control conditions for preparing hydrogels via ECM are well known to those skilled in the art, such as the decellularized extracellular matrix hydrogel of porcine dental pulp tissue and its preparation method disclosed in Chinese patent application document CN117258047A.

[0053] The term Matrigel, as used in this invention, refers to a soluble basement membrane matrix extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is a widely used ECM product in life science research, particularly as the gold standard for organoid culture. Currently a commercial product of Corning Incorporated, its main components include laminin, type IV collagen, nestin, heparin sulfate proteoglycan, and various growth factors.

[0054] The mechanical properties of the hydrogel referred to in this invention include viscosity, strain modulus, and dynamic modulus, which can be measured using a rheometer as known to those skilled in the art. Viscosity is measured by applying a constant shear rate or shear stress in steady-state shear mode, recording the corresponding stress and rate, and calculating viscosity as viscosity = shear stress / shear rate. Strain modulus is measured by applying slowly increasing shear strain to the sample in static or quasi-static mode, recording the stress response, and calculating strain modulus as strain = stress / strain. Dynamic modulus is measured by applying sinusoidal alternating shear stress / strain in oscillating shear mode, decomposing the response signal into storage modulus (i.e., G', elastic contribution) and loss modulus (i.e., G'', viscous contribution), which are collectively referred to as dynamic modulus. When G' > G'', the sample exhibits elasticity (more like a solid sample), and when G'' > G', it exhibits viscousity (more like a liquid sample).

[0055] The 4D Fast DIA proteomics research described in this invention is a new generation of protein quantification technology based on mass spectrometry. It integrates the ion mobility separation advantages of 4D proteomics with the unbiased detection characteristics of data-independent acquisition technology, and has wide applications in scientific research and clinical fields. Its basic operation is well known to those skilled in the art. Its experimental process is mainly divided into two core stages: library construction and detection analysis. In the sample pretreatment and library construction stage, proteins are first extracted from the sample, and peptides are obtained through enzymatic hydrolysis. Then, the sample composition is optimized through steps such as liquid chromatography fractionation. Subsequently, data is collected and a dedicated database is constructed. In the 4DFastDIA data acquisition stage, the pretreated peptide sample is injected into a Bruker timsTOFPro / Pro2 mass spectrometer. The instrument cyclically scans all ions within different mass-to-charge ratio windows, and simultaneously records four-dimensional data of retention time, mass-to-charge ratio, ion intensity, and ion mobility. In the data analysis and validation stage, the collected data is retrieved using the dedicated database to complete the qualitative analysis of peptides and proteins. Then, differential protein information is obtained through quality control analysis, quantitative variance statistics, etc.

[0056] Unless otherwise specified in this invention, any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose may be used. Even if the technologies, instruments, equipment, and materials used in this invention are specifically specified, it does not mean that this invention can only use these technologies, instruments, equipment, and materials, but merely represents the preferred solution of this invention. Those skilled in the art can still use any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose.

[0057] Example 1: Preparation method of SPF porcine skin ECM hydrogel

[0058] This invention provides a method for preparing SPF porcine skin ECM hydrogel, the method comprising:

[0059] Step 1, Sample Collection: After collecting SPF adult pig skin, remove the surface hair, fat layer and epidermis, clean it, cut it into 3.5 mm × 3.5 mm pieces, and freeze it at -80℃.

[0060] Step 2, Thawing: Take 50g of pig skin and thaw it quickly at 37℃.

[0061] Step 3, Degreasing: Use 1 L of 5% Na2CO3 as the degreasing solution, stir continuously at 35℃ and 300 rpm for 3 hours, and replace the degreasing solution with an equal amount every hour.

[0062] Step 4, Cleaning: Wash with pure water for 15 minutes at a time, stirring continuously, until the washing solution is neutral. Use absorbent paper to dry the surface moisture of the pigskin.

[0063] Step 5, pulping: Place the pigskin in 1 L 1× PBS, coarsely pulp it with a pulper, then homogenize it in an ice bath at 10000 rpm for 2 min. After homogenization, centrifuge the sample at 6000 rpm for 10 min and discard the supernatant.

[0064] Step 6, freeze drying: freeze-dry the precipitate at -60℃ and 1 Pa for 72 h.

[0065] Step 7, Grinding: Grind the freeze-dried sample into powder using a grinder. The powder can be stored at 4℃ for later use.

[0066] Step 8, Sterilization: Weigh 400 mg of sample and sterilize it at room temperature for 2 h with 40 mL of 0.1% peracetic acid + 4% anhydrous ethanol.

[0067] Step 9, Cleaning: Wash with an equal volume of physiological saline solution for 15 minutes each time, for a total of 4 washes, until the disinfectant solution is completely removed. Wash once with sterile pure water to remove salt. Centrifugation parameters during the cleaning process are: room temperature, 7200 g, 10 min.

[0068] Step 10, Enzymatic hydrolysis: Prepare 4 mg / mL pepsin with 0.01M HCl. After the enzyme is completely dissolved, filter it through a 0.22 µm filter membrane for sterilization and set aside. Hydrolyze the sample with 0.01M HCl and 1 mg / mL pepsin for 48 h. After hydrolysis, weigh 7200 g of the sample, centrifuge for 10 min, and collect the supernatant.

[0069] Step 11: Adjust pH and osmotic pressure: Add 1 / 100 of 1M NaOH and 1 / 9 of 10X PBS to make the hydrogel pH neutral and the osmotic pressure isotonic with that of the cells.

[0070] Step 12, aliquoting and storage: After aliquoting the hydrogel sample, store it at -20℃.

[0071] The final SPF pigskin ECM hydrogel product after gelation is shown in the image below. Figure 1 As shown.

[0072] Unless otherwise specified, step 5, homogenization, is included in all cases. However, if it is specifically stated that the hydrogel sample was obtained without homogenization, this step is not included. All other steps remain unchanged.

[0073] Unless otherwise specified, the raw materials used in this invention are all pigskins from adult SPF pigs, and in the case of studying young pigs, pigskins from 70-day-old SPF pigs are specifically used.

[0074] Example 2: Performance Experiment of the SPF Porcine Skin ECM Hydrogel of the Present Invention

[0075] 2.1 DNA Residue Detection

[0076] The DNA content in the adult pigs obtained from steps 1-2 of Example 1 of this invention and the adult pig ECMs after treatments 1-7 were detected by conventional agarose gel electrophoresis.

[0077] Figure 2 The diagram shows the DNA content detection results during the pig skin ECM processing of this invention. Lane 2 on the left is the marker lane, lanes 3-5 are the DNA content detection results of the raw adult pigs, and lanes 6-8 are the DNA content detection results of the adult pig ECM.

[0078] The test results showed that no DNA residue was found in the pig skin ECM of Example 1 of the present invention.

[0079] 2.2 Hydrogel water content detection

[0080] First, take an appropriate amount of cryovials, label them, and weigh them to obtain the "empty tube weight". Then, add about 1 mL of the final hydrogel sample obtained in Example 1 to the cryovials, incubate at 37°C for 30 min, weigh the gel-forming sample to obtain the "weight after gelation", and subtract the empty tube weight from this weight to obtain the "weight before lyophilization". Transfer the gel-forming sample to a -80°C freezer, pre-freeze and then lyophilize for 72 h, weigh it to obtain the "total weight after lyophilization", subtract the empty tube weight from this weight to obtain the "weight after lyophilization", and calculate the water content of the hydrogel sample (water content of hydrogel (%) = (weight before lyophilization - weight after lyophilization) ÷ weight before lyophilization × 100%).

[0081] Table 1 shows the water content of the porcine skin ECM hydrogel obtained in Example 1 of the present invention. Among them, adult-1 to adult-6 represent six different adult porcine skin ECM hydrogel samples, and 70-1 to 70-6 represent six different 70-day-old (juvenile) porcine skin ECM hydrogel samples.

[0082] Table 1. Water content of the porcine skin ECM hydrogel of the present invention

[0083]

[0084] The results in Table 1 show that there was no significant difference in water content between the ECM hydrogels obtained from adult pig skin and 70-day-old pig (juvenile) pig skin in this invention.

[0085] 2.3 Testing of the mechanical properties of hydrogels

[0086] The mechanical properties of the porcine skin ECM hydrogel sample obtained in Example 1 of this invention were tested using a rheometer, including viscosity, strain modulus, and dynamic modulus. Viscosity was measured by applying a constant shear rate or shear stress in steady-state shear mode, recording the corresponding stress and rate, and calculating viscosity as viscosity = shear stress / shear rate. Strain modulus was measured by applying slowly increasing shear strain to the sample in static or quasi-static mode, recording the stress response, and calculating strain modulus as strain = stress / strain. Dynamic modulus was measured by applying sinusoidally alternating shear stress / strain in oscillating shear mode, decomposing the response signal into storage modulus (G', elastic contribution) and loss modulus (G'', viscous contribution), which are collectively referred to as dynamic modulus. When G' > G'', the sample exhibits elasticity (more like a solid sample), and when G'' > G', it exhibits viscousity (more like a liquid sample).

[0087] Figure 3 shows the mechanical properties of the porcine skin ECM hydrogel of the present invention. The experimental groups included adult porcine skin ECM hydrogel (red dotted line) and 70-day-old porcine skin ECM hydrogel (blue dotted line). The measured parameters included viscosity (…). Figure 3a ), strain modulus ( Figure 3band Figure 3c ) and dynamic modulus ( Figure 3d and Figure 3e ).

[0088] Figure 3 shows that the viscosity of adult pig skin ECM hydrogel is greater than that of 70-day-old pig skin ECM hydrogel; the viscosity of the hydrogel remains stable within a strain range of 0.1%-10%; and G′ is higher than G″ in the frequency range of 0.1%-10%Hz, indicating the formation of an elastic hydrogel network.

[0089] 2.4 Scanning electron microscopy of hydrogels

[0090] After slicing the ECM hydrogel obtained in Example 1, the samples were adhered to the sample stage with conductive adhesive and subjected to gold sputtering using an ion sputtering instrument. Images were then acquired using a scanning electron microscope. The hydrogel slicing process employed both transverse and vertical cutting operations. Transverse cutting involved cutting perpendicularly along the hydrogel's thickness direction to obtain a cross-section with parallel top and bottom faces. A cryostat was used to ensure a smooth, tear-free cut. Vertical cutting involved cutting parallel along the length or width of the hydrogel to obtain a cross-section parallel to the surface. During cutting, the sample had to be aligned with a predetermined direction to avoid orientation deviations. Transverse cutting allows observation of the hydrogel's internal pore structure and cross-linking density distribution, verifying the material's internal homogeneity. Vertical cutting allows analysis of the interface transition zone between the surface and interior, evaluating the material's performance-related structures in the depth direction.

[0091] Figure 4 shows a scanning electron microscope image of the porcine skin ECM hydrogel of the present invention. The experimental group includes a heterogeneous group of adult porcine skin ECM hydrogels (i.e., hydrogels prepared without homogenization). Figure 4a For vertical cutting, Figure 4b (for cross-section processing) and homogenization group (i.e., homogenized during hydrogel preparation). Figure 4c It is a vertical cut. Figure 4d (It is a cross-section).

[0092] The scanning electron microscopy results in Figure 4 show that, regardless of whether the adult pig skin ECM hydrogel is homogeneous or not, the spatial structure of both transverse and vertical sections exhibits a network structure.

[0093] Figure 5 shows the frequency counts obtained from scanning electron microscopy and the pore size statistics fitted using Gaussian function modeling, describing the probability distribution of pore size using normal distribution characteristics. The fitted average diameter of the hydrogel prepared from the non-homogenized sample is 74.62 ± 37.75 μm. Figure 5a The fitted average diameter of the hydrogel prepared from the homogenized sample was 85.06 ± 44.16 μm. Figure 5b The difference is not significant.

[0094] 2.5 Hydrogel Proteomics Research

[0095] The proteomics of the hydrogel obtained in Example 1 of this invention was studied using 4D Fast DIA proteomics.

[0096] Tables 2 and 3 show the proteomics data of the porcine skin ECM hydrogel samples finally obtained in Example 1 of this invention. The experimental groups include adult porcine skin ECM hydrogels with and without homogenization (the left side of Table 2 shows the analysis results of the hydrogels without homogenization, and the right side shows the analysis results of the hydrogels after homogenization); and analysis results of adult and young porcine skin ECM hydrogels (the left side of Table 3 shows the analysis results of adult pigs, and the right side shows the analysis results of 70-day-old piglets).

[0097] The analysis results in Tables 2 and 3 show that the top 10 proteins in terms of strength are consistent between the homogenized and non-homogenized hydrogel samples, with only slight differences in the order of the last 4 proteins. Nine of the top 10 proteins in terms of strength are the same between adult and juvenile samples, with only slight differences in their ranking. These different proteins are also associated with the ECM or cytoskeleton, with the differentially expressed proteins highlighted in yellow.

[0098] Table 2. Comparative proteomics data between homogenized and unhomogenized samples.

[0099]

[0100] Table 3. Comparative proteomics data between adult and juvenile samples.

[0101]

[0102] Example 2: Mouse intestinal organoid culture

[0103] Figures 6-9 The results of organoid culture studies using different positive controls and porcine skin ECM hydrogel samples with different SPF values ​​obtained in Example 1 of this invention are shown. The organoid culture method is as follows: after resuspending the crypts in hydrogel, an appropriate amount of the suspension is spotted into wells of a plate, incubated at 37°C for 30 min, then organoid culture medium (Qiyuan, catalog number UC001) is added, and the plate is incubated at 37°C in a 5% CO2 incubator for observation.

[0104] 1) Preparation of crypt-hydrogel suspension

[0105] Add pre-cooled hydrogel solution to the purified crypt precipitate, gently pipette to mix, and resuspend until the density is 70 to 100 crypts per 10 μL matrix suspension. Spot the solution into the corresponding wells of the plate according to the recommended volume (50 µL for 24-well plates, 30 µL for 48-well plates). Incubate upside down in a 37°C, 5% CO2 incubator for 30 min to allow the hydrogel to completely solidify and form a three-dimensional scaffold supporting crypt growth.

[0106] 2) Culture medium addition and culture condition control

[0107] After the hydrogel solidifies, slowly add organoid culture medium along the well wall (500 µL for 24-well plates, 300 µL for 48-well plates) to avoid disrupting the hydrogel structure. Incubator parameters are strictly controlled at 37℃, CO2 concentration of 5%, and humidity ≥95%, with the culture medium changed every 2-3 days.

[0108] 3) Daily observation and recording

[0109] Daily observations were conducted using an inverted microscope to record crypt adhesion, proliferation, and the size and morphology of the organoids. Microscopic photographs were taken for archiving, and the organoid growth curve was tracked. Morphological identification involved observing the lacunar structure and epithelial cell arrangement of the organoids using HE staining to verify whether they mimicked in vivo tissue morphology. Biomarker detection employed immunofluorescence staining (Lgr5+, ZO-1, β-Cadherin) to detect intestinal stem cell markers and differentiation markers, confirming the functional characteristics of the organoids.

[0110] Figure 6 The results of organoid culture experiments over two days are shown, using the SPF porcine skin ECM hydrogel obtained in Example 1 of the present invention at a low concentration of 5 mg / mL, the SPF porcine skin ECM hydrogel obtained in Example 1 of the present invention at a high concentration of 10 mg / mL, and Matrigel (Corning) at a concentration of 8-10 mg / mL as a positive control. The figures show photographs of day 1 (left column) and day 2 (right column), respectively.

[0111] Figure 6 The results demonstrate that the porcine skin ECM hydrogel of this invention at various concentrations can support organoid growth.

[0112] Figure 7 The results of organoid culture experiments conducted over 3 days using 10 mg / mL of the final adult SPF porcine skin ECM hydrogel obtained in Example 1 of the present invention, 10 mg / mL of the final 70-day-old juvenile SPF porcine skin ECM hydrogel obtained in Example 1 of the present invention, and Matrigel (Corning Company) with a concentration of 8-10 mg / mL as a positive control are shown in the figures at 1 h, 24 h, and 72 h.

[0113] Figure 8 The images show the same experimental group after immunofluorescence staining (Lgr5+, ZO-1, b-Cadherin).

[0114] Figure 7 and Figure 8The results demonstrate that the SPF porcine skin ECM hydrogel of this invention, from different age sources, can support the growth of intestinal organoids and achieve organoid culture effects comparable to those of commercially available similar products.

[0115] Figure 9 The results show a comparison of organoid culture numbers. Generally, lower hydrogel concentrations result in better organoid culture, but excessively low concentrations can lead to insufficient support. The adult SPF porcine skin ECM hydrogel obtained in Example 1 of this invention was further diluted to study better organoid culture results while meeting support requirements. The average number of organoids per field of view in different gel groups was statistically analyzed. It was found that the SPF porcine skin ECM hydrogel of this invention, diluted to 3 mg / mL, still met the support requirements, and the number of organoids cultured was significantly higher than that cultured at commonly used concentrations of Matrigel (considered the gold standard for organoid culture in the industry), with 141 organoids in the former and 113 in the latter.

[0116] The above experimental results demonstrate that this invention, using SPF pigs as the target organism, has developed a high-purity, safe, and high-performance SPF pig skin ECM hydrogel product with promising commercial applications. This product can solve problems associated with existing hydrogel products such as Matrigel, including issues related to tumor origin, batch-to-batch variation, high cost, and safety concerns. Utilizing SPF experimental pig resources for the development of porcine tissue ECM hydrogels simultaneously promotes the high-value transformation of pig resources from traditional animal husbandry to the fields of bio-interdisciplinary collaboration and biomedicine.

[0117] The above descriptions are merely embodiments of the present invention. Commonly known technical knowledge in the solutions is not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the filing date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical well-known technologies should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing an SPF porcine skin extracellular matrix (ECM) hydrogel, characterized in that, Including the following steps: S1: Collect SPF pigskin and pre-process it; S2: Degrease the pretreated SPF pigskin; S3: Enzymatic hydrolysis of freeze-dried powder after sterilization and disinfection; S4: Adjust the pH and osmotic pressure of the supernatant after enzymatic hydrolysis to obtain the hydrogel product.

2. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis described in step S3 is carried out using 0.05-4 mg / ml pepsin in 0.005-0.1 M hydrochloric acid.

3. The preparation method according to claim 1 or 2, characterized in that, In step S4, pH and osmotic pressure are adjusted using NaOH and PBS to make the hydrogel pH neutral and the osmotic pressure isotonic with that of the cells.

4. The preparation method according to any one of claims 1-3, characterized in that, The degreasing process described in step S2 uses Na2CO3 as the degreasing solution, and is continuously stirred at 30-40℃ with a stirring speed of 300-800 rpm for 2-6 hours, replacing an equal amount of the degreasing solution every hour.

5. The preparation method according to any one of claims 1-4, characterized in that, The process includes step S2-1 after step S2: homogenizing the defatted pigskin, freeze-drying the precipitate and grinding it into powder; the homogenization process includes placing the pigskin in PBS, coarsely beating it with a pulper, homogenizing it in an ice bath at 8000-12000 rpm for 1-5 min, centrifuging the homogenized sample at 4000-10000 rpm for 5-30 min, and discarding the supernatant.

6. The preparation method according to any one of claims 1-5, characterized in that, Specifically, the following steps are included: S1: After collecting SPF adult pig skin, remove the surface hair, fat layer and epidermis, clean it, and cut it into 3.5mm×3.5mm small pieces; S2: Use 1 / 20 (W / V) 5% Na2CO3 as the degreasing solution, stir continuously at 35℃ and 300 rpm for 3 hours, and replace the degreasing solution with an equal amount every hour; wash the degreased pigskin with pure water for 15 minutes at a time, stirring continuously, until the washing solution is neutral, and then use absorbent paper to dry the surface moisture of the pigskin. S2-1: Place the defatted pigskin in 1 / 20 (W / V) 1× PBS, coarsely beat it with a homogenizer, then homogenize it at 10000 rpm for 2 min in an ice bath. After homogenization, centrifuge the sample at 6000 rpm for 10 min and discard the supernatant. Freeze-dry the precipitate at -60℃ and below under 1 Pa conditions for 72 h. Grind the freeze-dried sample into powder using a grinder and store it at 4℃ for later use. S3: Weigh 400 mg of lyophilized powder sample and sterilize it at room temperature for 2 h with 40 mL of 0.1% peracetic acid + 4% anhydrous ethanol. Wash with the same volume of physiological saline as the disinfectant solution for 15 min each time, for a total of 4 washes, until the disinfectant solution is completely removed; wash once with sterile pure water to remove salt; the centrifugation parameters during the washing process are room temperature, 7200 g, 10 min; prepare 4 mg / mL pepsin with 0.01M HCl, and after the enzyme is completely dissolved, filter it through a 0.22 µm filter membrane for sterilization before use; enzymatically digest the sterilized and washed lyophilized powder sample with 0.01M HCl and 1 mg / mL pepsin for 48 h, centrifuge the digested sample at 7200 g for 10 min, and collect the supernatant; S4: Add 1 / 100 of 1M NaOH and 1 / 9 of 10X PBS to make the hydrogel pH neutral and the osmotic pressure isotonic with that of the cells; after aliquoting the obtained hydrogel sample, store it at -20℃.

7. The SPF porcine skin ECM hydrogel prepared by the preparation method according to any one of claims 1-6.

8. The application of the SPF porcine skin ECM hydrogel of claim 7 in three-dimensional cell culture.

9. The application according to claim 8, characterized in that, The application is in organoid culture.

10. The application according to claim 9, characterized in that, The organoids mentioned are intestinal organoids.

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