Matrix scaffold for cell amplification and application

Abstract

The invention relates to a matrix scaffold for cell expansion and application. The matrix scaffold is prepared by the following steps: acquiring lymph nodes, and removing residual fat and blood cells to obtain primarily treated lymph nodes; grinding the primarily treated lymph nodes to obtain primarily ground lymph nodes; transferring the primarily ground lymph nodes into a cell screen, and continuously grinding to obtain a film-shaped acellular extracellular matrix material; enzymatically hydrolyzing the film-like acellular extracellular matrix material to remove residual cellular components; and carrying out sterilization treatment on the film-shaped acellular extracellular matrix material after enzymolysis to obtain the acellular extracellular matrix scaffold. The prepared pig lymph node acellular matrix scaffold has the advantages of being small in cell residual risk, low in immunogenicity, capable of retaining tissue specificity and structural characteristics, capable of amplifying human immune cells and the like, and has good application prospects in the fields of human immune cell in-vitro amplification, organ regeneration and the like.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedical materials, tissue engineering and regenerative medicine, and in particular to a matrix scaffold for cell expansion and its application, specifically to a decellularized extracellular matrix scaffold based on porcine lymph nodes and its application in the expansion of human immune cells. Background Art

[0002] As the leading cause of death globally, cancer treatment highly relies on the innovation of immunotherapy. As a powerful tool for cancer treatment, activated T cells and B cells with antibody-secreting functions play important roles in immunotherapy, bringing new hope for anti-tumor treatment. The core challenge of adoptive T cell therapy lies in how to achieve efficient activation and functional expansion of T cells. The current mainstream clinical protocols rely on CD3 / CD28 antibody-conjugated magnetic beads or cytokine stimulation such as IL-2, but there are significant limitations. For example, there are problems such as a long activation and expansion cycle of T cells, low activation efficiency of T cells, and unsatisfactory T cell function (non-targeted activation is likely to induce T cell clonal chaos, reducing treatment precision). Similar problems exist in the in vitro functional reconstruction of B cells. Activated B cells are usually few in number, difficult to isolate, and prone to apoptosis without immune cell support. The differentiation of B cells into plasma cells or memory B cells depends on the spatio-temporal co-regulation of signal networks such as follicular helper T cells (Tfh), follicular dendritic cells (FDC), and CXCL13 / IL-21 in the GC microenvironment, while existing in vitro culture systems (such as Transwell co-culture, 3D hydrogels) can only partially simulate single-factor stimulation and cannot reproduce the dynamic reaction characteristics of the GC. That is, the existing technology cannot generate GC-like B cells in vitro and control the kinetics of the GC-like reaction. Therefore, constructing an in vitro biomimetic microenvironment to simultaneously achieve efficient activation, functional expansion, and phenotypic homeostasis maintenance of T / B cells is a difficult problem for promoting the leap of immunotherapy towards clinical-grade standardized production.

[0003] Currently, decellularized extracellular matrix (dECM) has been applied to tissue engineering such as skin, bone, heart, and kidney. Badylak et al. reported in 1995 the use of a decellularized scaffold of small intestinal submucosa (SIS) for Achilles tendon repair and demonstrated the application potential of dECM in repairing soft tissue injuries. Ott et al. reported in 2008 a dECM scaffold of the entire rat heart prepared by perfusion decellularization technology, which achieved a breakthrough application of dECM in cardiac tissue engineering and marked an important milestone in this field. Jang et al. used a cardiac tissue-derived dECM bioink loaded with stem cells for 3D cell printing in 2017, improving the interaction and differentiation ability between stem cells.

[0004] Studies have shown that pigs and humans share many similar genes (the similarity between humans and pigs in certain genes is as high as 97.7%). Pig organs are similar to human organs in size, structure, and function, and their physiological and anatomical structures are highly similar to those of humans. As an important part of the organism's immune system, lymph nodes have functions such as filtering lymph fluid, clearing pathogens, generating immune responses, and producing antibodies. Pig lymph nodes contain abundant biochemical signals and immune regulatory factors. Compared with human lymph nodes limited by the scarcity of donors and ethical review barriers, pig lymph nodes have become an ideal source of biological materials for constructing acellular lymph node matrices due to their large-scale supply capacity.

[0005] Due to differences in composition among different tissues, different decellularization methods may cause varying degrees of damage to the structure and biological activity of the extracellular matrix, especially for thick tissues or organs with complex vascular structures. Therefore, the decellularization technique has a significant tissue dependence on the structural integrity of the extracellular matrix and the retention rate of bioactive molecules. For the acellular extracellular matrix of pig lymph nodes, there is currently no effective technical method to ensure that the cellular components in the matrix are removed while retaining tissue specificity and structural characteristics.

[0006] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, when the applicant made this invention, a large number of literatures and patents were studied, but all the details and contents were not listed in detail due to space limitations. However, this does not mean that this invention does not possess the features of these prior arts. On the contrary, this invention already possesses all the features of the prior arts, and the applicant reserves the right to add relevant prior arts in the background art. Summary of the Invention

[0007] Based on the great potential of the acellular extracellular matrix scaffold of pig lymph nodes for expanding human immune cells, but there is currently no work report on the acellular extracellular matrix derived from pig lymph nodes. A small risk of cell residue and the ability to retain tissue specificity and structural characteristics play an important role in realizing the expansion of human immune cells and organ regeneration based on the acellular extracellular matrix scaffold of pig lymph nodes. Therefore, it is necessary to develop a method for preparing acellular extracellular matrix from pig lymph nodes using decellularization technology and for in vitro expansion and culture of human immune cells.

[0008] Aiming at the deficiencies of the prior art, the first aspect of the present invention provides a matrix scaffold for cell expansion, which is prepared by the following steps:

[0009] Obtain lymph nodes, remove residual fat and blood cells to obtain preliminarily processed lymph nodes;

[0010] Grind the preliminarily processed lymph nodes to obtain preliminarily ground lymph nodes;

[0011] Transfer the preliminarily ground lymph nodes to a cell sieve and continue grinding to obtain a thin-film-like acellular extracellular matrix material;

[0012] Digest the thin-film-like acellular extracellular matrix material with enzymes to remove residual cell components;

[0013] Sterilize the enzyme-digested thin-film-like acellular extracellular matrix material to obtain an acellular extracellular matrix scaffold.

[0014] According to a preferred embodiment, the lymph nodes are porcine lymph nodes.

[0015] According to a preferred embodiment, during the grinding process, the lymph nodes are rinsed with PBS solution containing 1% penicillin-streptomycin double antibody.

[0016] According to a preferred embodiment, the preliminarily ground lymph nodes are transferred to a cell sieve and continue grinding to obtain a light yellow thin-film-like porcine lymph node acellular extracellular matrix material.

[0017] According to a preferred embodiment, the cell sieve is a 100μm cell sieve.

[0018] According to a preferred embodiment, trypsin is used in the enzyme digestion step.

[0019] According to a preferred embodiment, 0.05% trypsin is used in the enzyme digestion step.

[0020] According to a preferred embodiment, the enzyme digestion duration is 2 hours.

[0021] According to a preferred embodiment, the enzyme-digested thin-film-like acellular extracellular matrix material is sterilized to obtain a sterile milky white thin-film-like porcine lymph node acellular extracellular matrix scaffold.

[0022] According to a preferred embodiment, peracetic acid is used to sterilize the enzyme-digested thin-film-like acellular extracellular matrix material.

[0023] The second aspect of the present invention provides a method for preparing a matrix scaffold for cell expansion, comprising the following steps:

[0024] Obtain lymph nodes, remove residual fat and blood cells to obtain preliminarily processed lymph nodes;

[0025] Grind the preliminarily processed lymph nodes to obtain preliminarily ground lymph nodes;

[0026] Transfer the preliminarily ground lymph nodes to a cell sieve and continue grinding to obtain a thin-film-like acellular extracellular matrix material;

[0027] Digest the thin-film-like acellular extracellular matrix material with enzymes to remove residual cell components;

[0028] The enzymatically digested membranous acellular extracellular matrix material is sterilized to obtain an acellular extracellular matrix scaffold.

[0029] The third aspect of the present invention provides the application of the matrix scaffold provided by the first aspect of the present invention and the preparation method of the matrix scaffold provided by the second aspect of the present invention in the amplification of immune cells.

[0030] According to a preferred embodiment, the immune cells are human immune cells.

[0031] According to a preferred embodiment, the immune cells include T cells and / or B cells.

[0032] According to a preferred embodiment, when the matrix scaffold provided by the first aspect of the present invention is co-cultured with immune cells, the immune cells are inoculated at a density of 0.1 to 1×10 7 cells / mL.

[0033] The fourth aspect of the present invention provides the application of the matrix scaffold provided by the first aspect of the present invention and the preparation method of the matrix scaffold provided by the second aspect of the present invention in organ regeneration.

[0034] The fifth aspect of the present invention provides the application of the matrix scaffold provided by the first aspect of the present invention and the preparation method of the matrix scaffold provided by the second aspect of the present invention in the preparation of tissue engineering materials or repair materials.

[0035] The technical effects of the present invention:

[0036] Traditional chemical decellularization methods (such as SDS, Triton X-100, etc.) dissolve DNA and intracellular components by destroying cell membranes, but there are many problems. For example, it is difficult to completely remove the reagents embedded in the pores of the ECM (extracellular matrix), resulting in toxic residues; strong chemical effects degrade key structures such as collagen fibers and laminin, losing biomechanical strength; small molecules such as GAG (glycosaminoglycan) and growth factors are easily dissolved or denatured by chemical reagents. While the present invention uses physical grinding method to achieve decellularization, avoiding the residue of chemical reagents; the microfluidic flushing effect generated during the grinding process synchronously removes fragmented DNA and cytoplasmic residues, and the residual DNA content is significantly reduced; the natural three-dimensional topological structure of the ECM is retained; low immunogenicity. Therefore, the acellular extracellular matrix scaffold prepared by the physical grinding method can utilize the retained RGD and laminin and be applied to cell activation and amplification, and further applied to the treatment of immune diseases. In addition, it can also be applied to the construction of 3D lymph node organoid models. Description of the Drawings

[0037] Figure 1 It is a schematic diagram of the co-culture of a porcine lymph node acellular extracellular matrix scaffold and human immune cells;

[0038] Figure 2Results of the effects of different decellularized chemical reagents on the decellularization efficiency of lymph nodes;

[0039] Figure 3 Figure showing the results of the effect of the decellularized matrix scaffold after chemical reagent treatment on the proliferation of immune cells;

[0040] Figure 4 shows the decellularization efficiency results of lymph nodes treated by the pure physical grinding method;

[0041] Figure 5 Results of the proliferation of immune cells co-cultured with the decellularized matrix scaffold prepared by the pure physical grinding method within 12 days;

[0042] Figure 6 Results of the proteomics analysis of the decellularized matrix material prepared by the pure physical grinding method. Detailed implementation manners

[0043] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these descriptions are exemplary and are not intended to limit the scope of the present disclosure. In the following detailed description, for the sake of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. However, obviously, one or more embodiments can also be implemented without these specific details. In addition, in the following description, descriptions of well-known technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer are conventional products that can be obtained through commercial purchase. All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used here should be interpreted as having a meaning consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0044] According to the present invention, decellularized extracellular matrix (dECM) refers to a biomaterial formed by removing immunogenic components from human or animal tissues / organs / tissues through decellularization technology. The removal of immunogenic cellular components reduces potential adverse immune reactions. At the same time, this naturally sourced biomaterial itself is rich in extracellular macromolecular three-dimensional frameworks such as collagen, elastin, fibronectin, laminin, and matrix cell proteins, retaining the physicochemical signals and biological properties of the source tissue and being an ideal material for regenerating functional organs / tissues.

[0045] Example 1

[0046] This embodiment provides a preparation method of a matrix scaffold for cell expansion, and the matrix scaffold is particularly a decellularized extracellular matrix scaffold based on porcine lymph nodes.

[0047] According to this embodiment, the preparation method includes the following steps:

[0048] S1 Sample pretreatment

[0049] Take fresh porcine neck muscle tissue (pig trough head meat), use a sterile scalpel to cut the tissue into small pieces. When palpating the meat part with fingers and finding hard lumps or granular foreign bodies, use the scalpel to strip along the capsule to expose the grayish-white or light yellow lymph node tissue, and use sterile surgical scissors to completely strip the lymph nodes to obtain porcine lymph node tissue.

[0050] S2 Preliminary treatment of lymph nodes

[0051] Rinse the lymph nodes 3 times with sterile water to remove residual fat and blood cells; use surgical scissors to trim the fibrous membrane (outer white membrane) around the lymph nodes; then cut the lymph node tissue into homogeneous small pieces of 0.1 cm 3 and perform subsequent treatment according to the ratio of 10 g of tissue / 50 mL of solution after weighing.

[0052] S3 Decellularization treatment and drying

[0053] Enzymatic decellularization: Add 0.02% trypsin solution according to the density of 10 g of tissue / 50 mL of solution, stir for 24 hours, and then wash with deionized water for 5 minutes; then wash with 2×PBS buffer (pH 7.2) for 15 minutes to terminate the reaction.

[0054] Chemical decontamination treatment (performed in sequence, 2 hours for each step): Add 3% Tween, 102 mM SDC solution, and 0.1% peracetic acid to the enzymatically digested tissue in sequence for treatment. Finally, transfer the tissue to deionized water and wash for 24 h for thorough washing.

[0055] Drying: Place the treated tissue in a freeze dryer at -80 °C for 48 hours to obtain a white decellularized extracellular matrix scaffold (dECM).

[0056] Example 2

[0057] This embodiment provides the exploration results of co-culturing the decellularized extracellular matrix scaffold with human primary B cells, wherein the decellularized extracellular matrix scaffold is prepared by the preparation method of Example 1.

[0058] The steps of the co-culture experiment of the decellularized extracellular matrix scaffold with human primary B cells are as follows:

[0059] S1 Pretreatment of the decellularized extracellular matrix scaffold

[0060] The prepared acellular extracellular matrix scaffold of porcine lymph nodes was soaked in 0.1% peracetic acid solution for sterilization and then transferred to a sterile cell culture dish. It was soaked in RPMI-1640 medium for 24 hours to enhance the adhesion between the scaffold and the bottom of the culture dish and between the scaffold and the cells.

[0061] Inoculation and culture of S2 cells

[0062] Human primary B cells were processed to a density of 1×10 6 cells / mL (i.e., human primary B cells were resuspended in fresh medium at a density of 1×10 6 cells / mL), and then human primary B cells were co-cultured with the processed acellular extracellular matrix scaffold. As Figure 1 shown, after inoculating the cells, they were placed in a cell culture incubator and cultured for 45 minutes at 37°C and a carbon dioxide concentration of 5%. Then, medium was added and the culture continued.

[0063] According to this example, in the co-culture experiment of human primary B cells and the acellular extracellular matrix scaffold of the chemical reagent group, the grouping was set as follows: 1 control group and 3 parallel experimental groups (denoted as Material Group 1, Material Group 2, and Material Group 3 respectively). Among them, the control group was a group of human primary B cells alone without any scaffold material; the three parallel experimental groups were all groups of co-culturing human primary B cells with the acellular extracellular matrix scaffold of porcine lymph nodes treated by chemical reagent (SDC) for decellularization. The results of the co-culture experiment of human primary B cells and the acellular extracellular matrix scaffold are as Figure 3 shown. Figure A shows the bright-field microscope observation results of the co-culture of human primary B cells and the acellular extracellular matrix scaffold on the 3rd and 5th days. Among them, the scale bar = 100 μm; Figure B is a line graph of the quantitative cell viability by the CCK-8 method (absorbance detection at 450 nm). Among them, the abscissa represents different groups and the ordinate represents the OD value.

[0064] Figure 3 The results showed that: on the 3rd day of co-culture, cell colonies grew out significantly in the 3 experimental groups, and the proliferation effect was relatively obvious; but on the 5th day of culture, the cells in the 3 experimental groups all began to apoptose. In this result, the main reason for cell apoptosis was the residual toxicity of the chemical decellularization reagent, which inhibited the growth of B cells. That is, it was shown that the scaffold material treated with the chemical decellularization reagent (SDC) had a cytotoxic effect on human primary B cells, and starting from the 5th day, the cells in the experimental groups began to apoptose.

[0065] Example 3

[0066] This example provides a preparation method of acellular extracellular matrix of porcine lymph nodes based on physical grinding. Specifically, this example explores the effect of pure physical pre-grinding on the decellularization efficiency of porcine lymph nodes and the promotion of the proliferation of human primary B cells.

[0067] The preparation method of porcine lymph node acellular extracellular matrix based on physical grinding includes the following steps:

[0068] S1 Physical grinding for decellularization

[0069] Grinding: Place the small pieces of porcine lymph node tissue (about 0.1 cm 3 ) on a 100-mesh sieve for grinding. During the grinding process, use PBS solution containing double antibiotics (1% penicillin-streptomycin or denoted as pS) to rinse and grind at the same time until the material has no granularity, obtaining the preliminarily ground material; transfer the preliminarily ground material to a sterile 100-μm cell sieve and continue to grind in small amounts and multiple times. During this process, use PBS solution containing double antibiotics (1% penicillin-streptomycin or denoted as ps) to rinse several times until a light yellow film-like porcine lymph node acellular extracellular matrix scaffold is obtained.

[0070] S2 Trypsin (trypsin) digestion to remove residual cell components

[0071] Digestion conditions: Place the porcine lymph node acellular extracellular matrix scaffold obtained in step S1 at 37 °C, add 0.05% trypsin to it, and continuously digest for 2 hours to decompose the protein connections between cells and promote cell dissociation.

[0072] Sterilization treatment: Place the digested porcine lymph node acellular extracellular matrix scaffold in a 0.1% peracetic acid solution for disinfection and sterilization, and place it on a rotary shaker overnight. Rinse 3 - 5 times with PBS containing double antibiotics (ps) to obtain a sterile milky white film-like porcine lymph node acellular extracellular matrix scaffold.

[0073] Detection: In this example, DNA quantitative detection is carried out by Nanodrop; the amount and length of residual DNA in the porcine lymph node acellular extracellular matrix scaffold are determined by DNA gel electrophoresis; whether there are residual cell components in the porcine lymph node acellular extracellular matrix scaffold tissue is determined by hematoxylin-eosin (HE) staining.

[0074] Figure 2 The results of the effects of different decellularization chemical reagents on the decellularization efficiency of lymph nodes Figure 2Among them, Diagram A is a statistical chart of the DNA content remaining in the sample after treating lymph nodes with different decellularization chemical reagents; Diagram B is a gel electrophoresis diagram of the DNA content remaining in the sample after treating lymph nodes with different decellularization chemical reagents; Diagram C is the HE staining result of the sample after treating lymph nodes with different decellularization chemical reagents. The results of Diagrams A and B show that after treating lymph nodes with SDC, Triton-x100, and SDC+Triton-x100, the remaining DNA content in the SDC treatment group is significantly lower than that in other treatment groups, that is, the decellularization efficiency of the SDC treatment group for lymph nodes is the highest. The staining result of Diagram C matches Diagrams A and B. In Diagram C, the blue-stained cell nuclei in the control group are more distributed in the tissue. In the SDC group, the cell nuclei completely disappear, and the tissue forms a clear and regular reticular pore structure after decellularization.

[0075] Figure 4 shows the decellularization efficiency results of treating lymph nodes by the pure physical grinding method. Figure 4.1 Among them, Diagram A is the decellularization flow chart of the pure physical grinding method. After decellularization, the fresh tissue becomes a milky white film-like scaffold, which can be further formed into a gel after freeze-drying, grinding into powder, and digestion; Diagram B is a statistical chart of the DNA content remaining in the sample after treating lymph nodes by the pure physical grinding method; In collagen, the content of HYP is relatively stable and characteristic. Therefore, the content of collagen can be indirectly reflected by detecting the content of HYP. Diagram C shows the influence of different decellularization methods (SDC chemical decellularization method or pure physical grinding decellularization method) on the content of HYP (hydroxyproline) in the tissue. The results prove that during the decellularization process, while removing DNA substances such as cells, the pure physical grinding method can retain HYP (hydroxyproline), that is, collagen content, to the greatest extent. The collagen content retained in the physical grinding group is significantly higher than that in the SDC group; Diagram D is a gel electrophoresis diagram of the DNA content remaining in the sample after treating lymph nodes by the pure physical grinding method. This result shows that the pure physical grinding method can effectively remove cell components. Figure 4.2 Among them, the control group and the physical grinding group before and after decellularization were subjected to HE staining. The result of Diagram E shows that the cell nuclei in the physical grinding group were removed after decellularization treatment, and the decellularized extracellular matrix presented a loose and porous structure; The SEM images (Figure F) of the control group and the physical grinding group show that the tissue of the control group is denser and has smaller voids, while there are no obvious cell components in the electron micrograph of the physical grinding group, and the pores are larger and arranged regularly, presenting a three-dimensional reticular structure. Among them, the scale bar = 100μm. The results of Figure 4 show that the decellularization efficiency of treating lymph nodes by the pure physical grinding method is similar to that of the SDC treatment group, indicating that the pure physical grinding method can achieve efficient DNA removal. The remaining DNA content in the sample treated by the pure physical grinding method is about 66.75 ng / mg of tissue dry weight. Using the pure physical grinding method can also completely avoid the potential toxicity of chemical reagent residues.

[0076] Figure 5 The results of immune cell proliferation after co-culturing the acellular matrix scaffold prepared by the pure physical grinding method with immune cells within 12 days. The control group was the treatment without the acellular extracellular matrix scaffold, and Material Group 1, Material Group 2, and Material Group 3 were parallel experimental groups with the scaffold material added. Figure 5 Among them, Figure A and Figure B are the bright-field cell images and live-dead staining fluorescence images of the parallel experimental groups taken on the 12th day of co-culture. Among them, the scale bars in Figure A and B = 200 μm. Figure 5 The results showed that after co-culturing the scaffold material decellularized by the physical grinding method with human primary B cells, a large number of B cells were observed to adhere and grow towards the scaffold material. And on the 12th day of culture, live-dead fluorescence staining showed that most B cells had good activity (Figure B); the cells in the wells were counted every 3 days, and the B cell proliferation curve graph (Figure C) was drawn. The results showed that on the material, the B cell proliferation effects of the 3 parallel experimental groups were obvious, and they were amplified nearly 20 times within 12 days, which was better than the control group without the scaffold material.

[0077] Figure 6Results of mass spectrometry (MS) analysis of porcine lymph node acellular extracellular matrix materials. In this example, 480 proteins and 1683 peptide segments were identified. Further, its biological functions were inferred through bioinformatics analysis, and functional clustering analysis (including biological processes and molecular functions) was performed through Gene Ontology (GO). Figure A shows the results of protein abundance distribution. In this example, 480 proteins were sorted according to their relative expression abundances. The yellow area represents the protein range covering 90% of the total protein abundance. The results show that although the identified proteins have high diversity, the relative expression abundances of most proteins are less than 1% of the total protein amount. Multiple types of collagen are still the most abundant protein types, including collagen fibrils (types 1, 4, 6 and their subtypes). Figure B shows some key proteins sorted according to the percentage of relative protein abundance. The mainly highly expressed ones include multiple collagen subtypes such as COL1A1, COL4A1, COL4A2, COL5A2, COL6A1-6A3, and COL6A5. These collagens constitute the main structural components of the ECM and provide mechanical support; it also includes laminin subunits such as LAMA3, LAMA4, LAMA5, and LAMB2. This part of the proteins is involved in basement membrane formation and cell adhesion; IGHM (immunoglobulin M heavy chain) is related to the antibodies secreted by B cells; the high-abundance expressions of ITGB1, CD44, and FN1 are related to cell migration activities, and may be related to the processes of immune cell infiltration (such as T cells penetrating the ECM into the inflammatory site) and tumor metastasis (such as cancer cells invading through integrin-dependent pathways). In Figure C, the BP (biological process) analysis lists the key biological processes, most of which are related to the immune system, such as "positive regulation of T cell differentiation", "antigen processing and presentation", "activation of immune response", etc. In addition, there are also some proteins related to cell migration, cell adhesion, and extracellular matrix organization that are significantly expressed, indicating the presence of cell migration or organizational structure remodeling activities in the material.Panel D shows the MF (molecule function) molecular function analysis of the extracellular matrix proteins. Multiple functions related to the extracellular matrix (ECM), such as ECM structural constituents and binding (e.g., extracellular matrix structural constituent, collagen binding, ECM binding), ECM mechanical support (e.g., conferring compression / tensile strength), and integrin-mediated cell-matrix adhesion, etc., are significantly enriched, indicating the presence of matrix proteins related to extracellular matrix remodeling activities, tissue repair, fibrosis, or matrix remodeling in the tumor microenvironment. MHC protein binding is also a significant item, referring to antigen presentation in the immune system and being related to immune cell activation or adaptive immune responses. The proteomics results show that this dECM scaffold material is rich in protein components related to immune cell activation, is expected to support the culture of human primary immune cells, and plays an important role in intercellular signal transduction and tissue regeneration.

[0078] It should be noted that the above specific embodiments are exemplary. Those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also fall within the scope of the disclosure of the present invention and within the protection scope of the present invention. Those skilled in the art should understand that the description and drawings of the present invention are illustrative and do not constitute a limitation to the claims. The protection scope of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts. For example, "according to a preferred embodiment" indicates that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications according to each inventive concept. Throughout the text, the features guided by "according to a preferred embodiment" are only optional and should not be understood as being necessarily provided. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A matrix scaffold for cell expansion, characterized in that, It is prepared by the following steps: Obtain lymph nodes, remove residual fat and blood cells to obtain preliminarily treated lymph nodes; Grind the preliminarily treated lymph nodes to obtain preliminarily ground lymph nodes; Transfer the preliminarily ground lymph nodes to a cell sieve and continue grinding to obtain a thin-film acellular extracellular matrix material; Enzymatically digest the thin-film acellular extracellular matrix material to remove residual cell components; Sterilize the enzymatically digested thin-film acellular extracellular matrix material to obtain an acellular extracellular matrix scaffold.

2. The matrix scaffold according to claim 1, characterized in that The lymph nodes are porcine lymph nodes.

3. The substrate scaffold according to claim 1 or 2, characterized in that, During the grinding process, rinse the lymph nodes with PBS solution containing 1% penicillin-streptomycin double antibody.

4. The matrix scaffold according to claim 2, wherein Transfer the preliminarily ground lymph nodes to a cell sieve and continue grinding to obtain a light yellow thin-film porcine lymph node acellular extracellular matrix material.

5. The matrix scaffold according to claim 1, wherein The cell sieve is a 100μm cell sieve.

6. The matrix scaffold according to claim 1, characterized in that, Trypsin is used in the enzymatic digestion step.

7. The matrix scaffold according to claim 6, characterized in that, 0.05% trypsin is used in the enzymatic digestion step.

8. The matrix scaffold according to claim 2, wherein, Sterilize the enzymatically digested thin-film acellular extracellular matrix material to obtain a sterile milky white thin-film porcine lymph node acellular extracellular matrix scaffold.

9. A method for preparing a matrix scaffold for cell expansion, characterized in that, It includes the following steps: Obtain lymph nodes, remove residual fat and blood cells to obtain preliminarily treated lymph nodes; Grind the preliminarily treated lymph nodes to obtain preliminarily ground lymph nodes; Transfer the preliminarily ground lymph nodes to a cell sieve and continue grinding to obtain a thin-film acellular extracellular matrix material; Enzymatically digest the thin-film acellular extracellular matrix material to remove residual cell components; Sterilize the enzymatically digested thin-film acellular extracellular matrix material to obtain an acellular extracellular matrix scaffold.

10. Application of the matrix scaffold according to any one of claims 1 to 8 and the preparation method of the matrix scaffold according to claim 9 in immune cell expansion.

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