A method for preparing a cell-derived extracellular matrix and a cell-derived extracellular matrix-coated cell culture plate and applications thereof

Abstract

The present application belongs to the field of biomedical technology, and particularly relates to a cell-derived extracellular matrix and a preparation method and application of a cell culture plate coated with the cell-derived extracellular matrix. The present application cultures human astrocytes in a cell culture plate, and makes the human astrocytes secrete extracellular matrix. After the human astrocytes are added into a cell lysate for cell removal treatment, nucleic acid fragmentation treatment is performed to obtain the cell-derived extracellular matrix. The present application has relatively simple operation, short time period, and can realize batch production of extracellular matrix without external virus carrying and stable components. The obtained cell-derived extracellular matrix is resistant to storage, has good stability, is highly similar to a natural brain environment when coated on a cell culture plate, has good biocompatibility with cells, can be applied to tissue repair and regeneration as an ideal biomedical material, can regulate the progress and efficiency of human dermal fibroblast HDF transdifferentiation into neurons, and can obtain in-vitro survival neurons.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing and applying cell-derived extracellular matrix and cell culture plates coated with cell-derived extracellular matrix. Background Technology

[0002] The extracellular matrix, composed of proteins and polysaccharides secreted by cells, is widely studied and applied in biomedicine, tissue engineering, and drug development due to its excellent biocompatibility and the presence of many functional active molecules. The extracellular matrix mainly includes tissue-derived extracellular matrix (T-ECM) and cell-derived extracellular matrix (C-ECM). C-ECM is an extracellular matrix obtained by culturing cells in vitro. Different types of cells can be cultured to obtain extracellular matrices for specific cell types according to application requirements, and the composition and function of C-ECM can be customized by controlling experimental conditions. Related research has demonstrated that C-ECM, as an extracellular matrix, has biological functions that mimic tissue-like microenvironments, providing physical support for cells and regulating cell behavior, thus playing an important role in tissue development. Therefore, C-ECM is increasingly being developed for applications in tissue engineering, regenerative medicine, and organoid culture.

[0003] However, current C-ECM preparation still faces certain limitations, primarily low decellularization efficiency and poor stability of active ingredients. It is difficult to establish a standardized process to ensure consistent quality and performance across different batches of C-ECM. For example, in C-ECM preparation, cells are typically seeded on two-dimensional surfaces such as culture flasks or dishes. Although TC treatment of the culture flask or dish surface can enhance cell adhesion and proliferation, the C-ECM adhering to the surface is prone to breakage and detachment during washing, cell removal, and storage, resulting in a limited quantity of effective C-ECM. Furthermore, in cell removal, traditional chemical methods use high concentrations of the anionic surfactant sodium dodecyl sulfate (SDS). While removing cells, this process damages C-ECM, causing the loss of active ingredients (such as fibronectin and laminin). Additionally, the removed cells release their internal nucleic acids; if not completely removed, residual DNA fragments may trigger immune responses or calcification. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing cell-derived extracellular matrix and cell culture plates coated with cell-derived extracellular matrix, as well as their applications. The resulting cell-derived extracellular matrix has good stability of its effective components, can simulate the in vitro microenvironment of neuronal transdifferentiation, improves transdifferentiation efficiency, and enhances the effectiveness of the cell-derived extracellular matrix.

[0005] This invention provides a method for preparing cell-derived extracellular matrix, comprising the following steps: After obtaining gelatin-coated cell culture plates, the gelatin in the gelatin-coated cell culture plates is cross-linked with glutaraldehyde to obtain pretreated cell culture plates. Human astrocytes were cultured using the pretreated cell culture plate. When the cell confluence was ≥100%, the cells were cultured in a medium containing ascorbic acid to obtain the cultured cells. The cultured cells were lysed using a lysis buffer, and after cell removal, nucleic acid fragmentation was performed to obtain cell-derived extracellular matrix, which was then coated onto the cell culture plate. The cell lysis buffer consists of NH4OH, 20% v / v Triton X-100, and PBS, with a volume ratio of NH4OH, 20% v / v Triton X-100, and PBS of 1:1.25:47.75.

[0006] Preferably, the gelatin is a 0.2% gelatin solution, and the coating temperature is 37°C for 1 hour. The glutaraldehyde is a 1% glutaraldehyde solution, and the cross-linking time is 30 min; The volume ratio of the gelatin solution to the glutaraldehyde solution is 8:5.

[0007] Preferably, the cell culture plate is a 24-well cell culture plate, and the seeding density of human astrocytes is 7 × 10⁻⁶. 4 One hole / hole.

[0008] Preferably, the concentration of ascorbic acid in the culture medium containing ascorbic acid is 50 μg / mL, and the culture time using the culture medium containing ascorbic acid is 7 days.

[0009] Preferably, the nucleic acid fragmentation is performed using DNase I solution.

[0010] Preferably, the human astrocytes include human astrocytes HA1800.

[0011] The present invention also provides cell-derived extracellular matrix and / or cell culture plates coated with cell-derived extracellular matrix obtained by the preparation method described above.

[0012] The present invention also provides the application of the cell-derived extracellular matrix and / or cell culture plates coated with cell-derived extracellular matrix described in the above technical solutions in one or more of the following: (1) Inducing human dermal fibroblasts to differentiate into neurons; (2) To prepare products for tissue repair and / or regeneration; (3) Organoid culture.

[0013] This invention also provides a method for inducing human dermal fibroblasts to differentiate into neurons, comprising the following steps: Human dermal fibroblasts were seeded using cell-derived extracellular matrix and / or cell culture plates coated with cell-derived extracellular matrix as described in the above technical solution. When the cell confluence was ≥80%, lentivirus and polybrene were used to knock down their cells. PTBP1 Expression was selected using hygromycin B to obtain knockdown samples. PTBP1 Human dermal fibroblasts; Knock down PTBP1 Human dermal fibroblasts were induced to differentiate into neurons.

[0014] Preferably, the seeding density of the human dermal fibroblasts is 3.5 × 10⁻⁶. 4 One per hole; The concentration of the polyaluminum glycoside is 0.01 mg / mL; The concentration of hygromycin B is 0.1 mg / mL; The culture medium used for inducing differentiation includes N3 medium, the components of which include DMEM / F12, penicillin, insulin, transferrin, progesterone, putrescine, and sodium selenite.

[0015] Beneficial effects: This invention utilizes gelatin-coated cell culture plates. After obtaining the gelatin-coated cell culture plates, the gelatin in the gelatin-coated cell culture plates is cross-linked with glutaraldehyde to obtain pretreated cell culture plates. Human astrocytes are cultured in the pretreated cell culture plates to induce the secretion of extracellular matrix. The human astrocytes are then added to cell lysis buffer for decellularization and followed by nucleic acid fragmentation to obtain cell-derived extracellular matrix. This invention is relatively simple to operate, has a short cycle time, and can achieve batch production of extracellular matrix without exogenous virus carriers and with stable components. The obtained extracellular matrix coated on the cell culture plates is highly similar to the natural brain environment and has good biocompatibility with cells. It can serve as an ideal biomedical material for tissue repair and regeneration, especially for the differentiation and culture of neurons. The cell-derived extracellular matrix obtained using the preparation method provided in this invention is storage-resistant, showing no degradation even after 7 days of storage at 4°C. After 7 days of storage in DPBS solution containing 1% penicillin-streptomycin antibiotics, the extracellular matrix remains sterile, forming a dynamic network scaffold structure that can mimic the brain microenvironment, and can be applied to in vitro transdifferentiation. Human dermal fibroblasts grow on this extracellular matrix, and after knockdown... PTBP1 During the subsequent transdifferentiation into neurons, the signal transduction pathways sense and regulate the cell-derived extracellular matrix microenvironment, thereby increasing the yield of neurons that differentiate and survive in vitro. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 A schematic diagram of the process for preparing cell culture plates coated with extracellular matrix; Figure 2 This is a graph showing the results of decellularization efficiency testing for different cell types in Example 1; the scale bar is 300 μm. Figure 3 This is an image showing the immunofluorescence staining results of different types of extracellular matrix type I collagen and fibronectin in Example 2; the scale bar is 50 μm. Figure 4 Bright-field and scanning electron microscope images of different types of extracellular matrix were used to test Example 2; the scale bar for the bright-field images was 300 μm, and the scale bar for the scanning electron microscope images was 20 μm. Figure 5 The results of the relative content detection of a specific protein in Matrigel in Test Example 2; Figure 6 The results of the relative content detection of a specific protein in the extracellular matrix HA1800 ECM of Test Example 2 were obtained. Figure 7The results of the relative content detection of a specific protein in the extracellular matrix HDF ECM of Example 2 were used to test the results. Figure 8 The results of the relative content detection of a specific protein in the extracellular matrix (HFSC ECM) of Example 2 were used to test the results. Figure 9 Figure 3 shows the results of SDS-PAGE component stability tests on different types of extracellular matrix in Example 3. Figure 10 Figure showing the sterility test results of different types of extracellular matrix stored for 7 days in Test Example 3; Figure 11 This is a test of DAPI staining results for cells cultured in different types of extracellular matrix-coated cell culture plates in Example 4; the scale bar is 300 μm; ns indicates no significant difference. Figure 12 This is to test the relative viability of cells cultured in different types of extracellular matrix-coated cell culture plates in Example 4; Figure 13 Immunostaining results of DAPI and Tuj1 on day 9 for neurons in test case 5; scale bar is 50 μm; Figure 14 Immunostaining results of DAPI and Tuj1 on day 14 for neurons in test case 5; scale bar is 50 μm; Figure 15 Immunostaining results of DAPI and Tuj1 on day 21 for neurons in test case 5; scale bar is 50 μm; Figure 16 Figure showing the comparison of Tuj1-positive cells in neurons of test case 5 on days 9, 14, and 21. ns indicates no significant difference. express P ≤0.0001. Detailed Implementation

[0018] This invention provides a method for preparing cell-derived extracellular matrix, comprising the following steps: After obtaining gelatin-coated cell culture plates, the gelatin in the gelatin-coated cell culture plates is cross-linked with glutaraldehyde to obtain pretreated cell culture plates. Human astrocytes were cultured using the pretreated cell culture plate. When the cell confluence was ≥100%, the cells were cultured in a medium containing ascorbic acid to obtain the cultured cells. The cultured cells were lysed using a lysis buffer, and after cell removal, nucleic acid fragmentation was performed to obtain cell-derived extracellular matrix, which was then coated onto the cell culture plate. The cell lysis buffer consists of NH4OH, 20% v / v Triton X-100, and PBS, with a volume ratio of NH4OH, 20% v / v Triton X-100, and PBS of 1:1.25:47.75.

[0019] This invention utilizes gelatin to coat cell culture plates, thereby obtaining gelatin-coated cell culture plates.

[0020] In one embodiment, the gelatin used in this invention is a 1% (w / v) gelatin solution; in another embodiment, the gelatin used in this invention is a 0.2% gelatin solution. In one embodiment, the coating temperature is 37°C; in another embodiment, the coating temperature is 37°C. In one embodiment, the coating time is 1 h; in another embodiment, the coating time is 1 h. In one embodiment, the coating volume of the gelatin solution is 800 μL / well. In one embodiment, the cell culture plate is a 24-well cell culture plate. Gelatin, as a denatured collagen, provides a more biomimetic environment closer to that in vivo by coating the cell culture plate, promoting cellular metabolic activity and C-ECM secretion. Furthermore, integrins in C-ECM can recognize and bind to cell adhesion sequences (RGD sequences) in gelatin, thereby enhancing the initial attachment and spreading of C-ECM on the culture plate. Therefore, the present invention utilizes gelatin-coated cell culture plates, which has the advantages of easy cell adhesion, good biocompatibility, and resistance to cell detachment.

[0021] After obtaining the gelatin-coated cell culture plate, the present invention crosslinks the gelatin in the gelatin-coated cell culture plate with glutaraldehyde to obtain a pretreated cell culture plate.

[0022] In one embodiment, the present invention mixes the gelatin-coated cell culture plate with glutaraldehyde, performs a first wash after crosslinking to quench residual glutaraldehyde, and a second wash to obtain a pretreated cell culture plate. In one embodiment, the glutaraldehyde used in the present invention is a 1% (v / v) gelatin solution; in another embodiment, the glutaraldehyde used in the present invention is a 1% glutaraldehyde solution. In another embodiment, the crosslinking time is 30 min. In one embodiment, the volume ratio of the gelatin solution to the glutaraldehyde solution is 8:5. In one embodiment, the amount of glutaraldehyde solution used is 500 μL / well. The present invention crosslinks gelatin with glutaraldehyde, a highly efficient chemical crosslinking agent that can form strong covalent bonds with gelatin molecules, connecting dispersed gelatin molecules to form a network structure. This significantly enhances the mechanical strength and stability of the gelatin layer, reduces the possibility of C-ECM detaching during cell culture and decellularization, and ensures stable adhesion to the surface of the culture plate.

[0023] In one embodiment, the present invention utilizes a glycine solution to quench residual glutaraldehyde. In one embodiment, the concentration of the glycine solution in the present invention is 1 M. In one embodiment, both the first and second washes in the present invention are performed with DPBS solution. In one embodiment, the first and second washes are performed 1 to 2 times each.

[0024] After obtaining the pretreated cell culture plate, the present invention uses the pretreated cell culture plate to culture human astrocytes. When the cell confluence is ≥100%, the cells are cultured in a medium containing ascorbic acid to obtain the cultured cells.

[0025] As one embodiment, the seeding density of the human astrocytes described in this invention is 7 × 10⁻⁶. 4 Cells / well. As one embodiment, the human astrocytes described in this invention include human astrocytes HA1800. This invention selects human astrocytes, which, compared to other cells (such as human dermal fibroblasts and human hair follicle stem cells), produce a cell-derived extracellular matrix that is more resistant to storage. After being stored at 4°C for 7 days, it does not degrade. After being stored in DPBS solution containing 1% penicillin-streptomycin for 7 days, the extracellular matrix remains sterile and exhibits good biocompatibility. This provides significant advantages for neuronal differentiation and survival, effectively improving neuronal differentiation efficiency and promoting the survival of induced differentiated neurons.

[0026] In one embodiment, the human astrocytes described in this invention are cryopreserved and then revived human astrocytes. In another embodiment, the cryopreserved and revived human astrocytes are subjected to adherent culture, digested, and then cultured in the pretreated cell culture plate. In another embodiment, the digestion in this invention uses 0.25% Trypsin-EDTA. In another embodiment, the digestion time is 1-2 minutes.

[0027] In one embodiment, the concentration of ascorbic acid in the culture medium containing ascorbic acid described in this invention is 50 μg / mL. In another embodiment, the culture time in the culture medium containing ascorbic acid described in this invention is 7 days. The addition of ascorbic acid in this invention promotes the formation of cell-derived extracellular radicals.

[0028] In one embodiment, the ascorbic acid-containing culture medium of the present invention includes DMEM / F12, fetal bovine serum, ascorbic acid, and a combination of antibiotics. In one embodiment, the combination of antibiotics is a mixture of penicillin and streptomycin, and the amount of the combination of antibiotics added is 1% (v / v) of the total volume of the ascorbic acid-containing culture medium. In one embodiment, the amount of fetal bovine serum added is 2-10% (v / v) of the total volume of the ascorbic acid-containing culture medium; in another embodiment, the amount of fetal bovine serum added is 4-8% (v / v) of the total volume of the ascorbic acid-containing culture medium; in yet another embodiment, the amount of fetal bovine serum added is 5-6% (v / v) of the total volume of the ascorbic acid-containing culture medium.

[0029] After obtaining the cultured cells, the present invention uses a lysis buffer to lyse the cultured cells, removes the cells, and performs nucleic acid fragmentation to obtain cell-derived extracellular matrix, which is then coated on the cell culture plate; the cell lysis buffer includes NH4OH and 20% v / v Triton X-100.

[0030] In one embodiment, the cell lysis buffer of the present invention comprises NH4OH, 20% v / v Triton X-100, and PBS, wherein the volume ratio of NH4OH, 20% v / v Triton X-100, and PBS is 1:1.25:47.75. The present invention utilizes NH4OH and Triton X-100 for lysis, optimizing the composition and ratio of the cell lysis buffer, and has the advantages of rapid cell lysis without damaging the effective components of the extracellular matrix.

[0031] In one embodiment, the present invention uses DNase I solution for nucleic acid fragmentation. In another embodiment, the concentration of the DNase I solution is 10 μg / mL. After cell removal, the decellularized C-ECM is incubated with DNase I solution for nucleic acid fragmentation, digesting the nucleic acid molecules released from the cells. This ensures that the final C-ECM does not contain DNA molecules from lysed cells, guaranteeing a cell-free C-ECM with stable components and properties, without affecting subsequent applications of the C-ECM.

[0032] The present invention also provides cell-derived extracellular matrix and / or cell culture plates coated with cell-derived extracellular matrix obtained by the preparation method described above.

[0033] The cell-derived extracellular matrix obtained by this invention is highly similar to the natural brain environment and has good biocompatibility with cells. It can simulate the in vitro microenvironment for neuronal transdifferentiation, greatly improving the efficiency of transdifferentiation. It can be used as an ideal biomedical material for tissue repair and regeneration, especially for the differentiation and culture of neurons.

[0034] In view of the role of the cell-derived extracellular matrix and / or cell culture plates coated with cell-derived extracellular matrix provided by the present invention, the application of the cell-derived extracellular matrix and / or cell culture plates coated with cell-derived extracellular matrix in one or more of the following is also within the scope of protection of the present invention: (1) inducing human dermal fibroblasts to differentiate into neurons; (2) preparing products for tissue repair and / or regeneration; (3) organoid culture.

[0035] This invention also provides a method for inducing human dermal fibroblasts to differentiate into neurons, comprising the following steps: Human dermal fibroblasts were seeded using cell-derived extracellular matrix and / or cell culture plates coated with cell-derived extracellular matrix as described in the above technical solution. When the cell confluence was ≥80%, lentivirus and polybrene were used to knock down their cells. PTBP1 Expression was selected using hygromycin B to obtain knockdown samples. PTBP1 Human dermal fibroblasts; knockdown of the cells PTBP1 Human dermal fibroblasts were induced to differentiate into neurons.

[0036] As one embodiment, the seeding density of the human dermal fibroblasts described in this invention is 3.5 × 10⁻⁶. 4 Cells / well. As one embodiment, the human dermal fibroblasts described in this invention are human dermal fibroblast HDF.

[0037] In one implementation method, this invention uses psPAX2 as the packaging plasmid, pMD2.G as the envelope plasmid, and shPTBP1 as the core plasmid to prepare knockdown... PTBP1 The expressed lentivirus, shPTBP1 core plasmid, is disclosed in the prior art: https: / / doi.org / 10.1038 / nn.4297. As one implementation, this invention knocks down... PTBP1 A mixture of expressed lentivirus and polybrene was used to infect human dermal fibroblasts (HDF) and knock down their expression. PTBP1 Expression. As one embodiment, the concentration of the polybrene in this invention is 0.01 mg / mL. This invention utilizes knockdown... PTBP1 Co-infection with expressed lentivirus and polybrene knocks down HDF in human dermal fibroblasts. PTBP1 Advantages of gene expression.

[0038] In one embodiment, the concentration of hygromycin B in this invention is 0.1 mg / mL. In another embodiment, the culture medium used for inducing differentiation in this invention includes N3 medium. Taking 50 mL as an example, the components of the N3 medium in this invention include 0.5 mL of penicillin-streptomycin, 125 μL of 10 mg / mL insulin, 50 μL of 50 mg / mL transferrin, 50 μL of 20 μM progesterone, 50 μL of 100 μM putrescine, 50 μL of 30 μM sodium selenite, and 49.5 mL of DMEM / F12. In another embodiment, the penicillin-streptomycin is a mixture of penicillin and streptomycin.

[0039] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for preparing and applying a cell-derived extracellular matrix and a cell culture plate coated with the cell-derived extracellular matrix, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0040] The human astrocytes HA1800 used in this embodiment of the invention were purchased from ScienCell, Inc. (catalog number 1800), human dermal fibroblasts HDF were purchased from the American Type Culture Collection (ATCC) (catalog number PCS-201-012), and human hair follicle stem cells HFSC were obtained from the Sihe Gene Research and Development Laboratory.

[0041] Example 1 according to Figure 1 The procedure shown illustrates the preparation of cell culture plates coated with extracellular matrix of human astrocytes. The specific steps are as follows: 1. Preparation of human astrocyte extracellular matrix HA1800 ECM (1) In a biosafety cabinet, take a 24-well cell culture plate, add 800 μL of 0.2% (w / v) gelatin solution to each well (preheated at 37°C beforehand), and place at 37°C for at least 1 h to form a gelatin coating.

[0042] (2) Discard the 0.2% (w / v) gelatin solution, add 1 mL of DPBS solution to wash, then add 500 μL of 1% glutaraldehyde, and place at room temperature for 30 min to allow cross-linking between gelatin and glutaraldehyde.

[0043] (3) Discard the 1% glutaraldehyde solution, add 1 mL of DPBS solution to wash, and then add 500 μL of 1 M glycine solution to quench the residual glutaraldehyde solution.

[0044] (4) Discard the 1 M glycine solution, add 1 mL DPBS solution to wash, and repeat twice. Keep the pretreated cell culture plate for later use.

[0045] (5) Human astrocytes HA1800 were revived and cultured on 10 cm cell culture dishes.

[0046] (6) Digest the cells from step 5, discard the culture medium, add 3 mL of DPBS solution to wash 2-3 times, then add 1 mL of Trypsin-EDTA, shake to mix and cover the cells, and place in a 37℃ incubator for digestion.

[0047] (7) Add 3 mL of 10% FBS cell culture medium to stop digestion, mix the cells with a pipette, transfer the cell resuspension to a 15 mL EP tube, and centrifuge at 1000 rpm for 3 min at 4°C.

[0048] (8) Discard the supernatant, resuspend the cells in 10% FBS cell culture medium, count the cells, and seed 7 × 10⁶ cells into each well of the pretreated cell culture plate. 4 Cells were cultured in a 5% CO2, 37°C incubator.

[0049] (9) After 12 h, when the cell confluence is greater than 100%, the cells aggregate into a dense monolayer. Add 50 mg / mL ascorbic acid to the culture medium to make the final concentration 50 μg / mL.

[0050] (10) Then replace the culture medium with fresh medium containing 50 μg / mL ascorbic acid every day, and repeat this step for 7 days to allow the cells to secrete extracellular matrix.

[0051] 2. Human astrocyte extracellular matrix HA1800 ECM cell culture plates that can simulate the brain microenvironment were prepared through decellularization treatment. (1) Seven days after the preparation of the extracellular matrix in step 1, the original culture medium was discarded by using a sterile disposable Pasteur pipette, and 0.5 mL of DPBS solution was added to wash the cells 2-3 times.

[0052] (2) Add 0.5 mL of preheated cell lysis buffer to lyse the cells, and shake gently to ensure that the cell lysis buffer fully covers the cell surface.

[0053] (3) Observe the cell lysis under a bright field microscope until no cells are seen and the surface shows a fibrous structure, then stop the lysis.

[0054] (4) Use a sterile disposable Pasteur pipette to remove the cell lysate and add 0.5 mL of DPBS solution to wash the cells twice.

[0055] (5) Add 10 μg / mL DNase I solution to remove residual cellular DNA and incubate at 37°C for 30 min.

[0056] (6) Use a sterile disposable Pasteur pipette to remove DNase I solution and add 0.5 mL of DPBS solution to wash the cells twice.

[0057] (7) Observe the integrity of the extracellular matrix on the surface of the cell culture plate under a bright field microscope. At this time, the extracellular matrix is ​​coated on the surface of the cell culture plate.

[0058] (8) Cell culture plates coated with extracellular matrix can be used directly, or 0.5 mL of DPBS solution containing 1% penicillin-streptomycin can be added and stored at 4°C for later use (use as soon as possible, and store within 7 days).

[0059] Example 2 according to Figure 1 The procedure shown illustrates the preparation of cell culture plates coated with extracellular matrix of human dermal fibroblasts. The specific steps are as follows: 1. Preparation of human dermal fibroblast extracellular matrix (HDF ECM): The specific steps are similar to those in Example 1, except that human astrocytes HA1800 are replaced with human dermal fibroblasts (HDF).

[0060] 2. Following step 2 of Example 1, prepare human dermal fibroblast extracellular matrix (HDF) ECM cell culture plates that can simulate the brain microenvironment.

[0061] Example 3 according to Figure 1 The procedure shown illustrates the preparation of cell culture plates coated with extracellular matrix for human hair follicle stem cells. The specific steps are as follows: 1. Preparation of human hair follicle stem cell extracellular matrix (HFSC) ECM: The specific steps are similar to those in Example 1, except that human astrocytes HA1800 are replaced with human hair follicle stem cells HFSC.

[0062] 2. Following step 2 of Example 1, prepare human hair follicle stem cell extracellular matrix (HFSCECM) cell culture plates that can simulate the brain microenvironment.

[0063] Test Example 1 1. Samples to be tested: Undecellularized extracellular matrix obtained in step 1 of Examples 1-3, and decellularized extracellular matrix obtained in step (7) of step 2 of Examples 1-3.

[0064] 2. The samples to be tested were stained with DAPI, and observed and photographed under a bright-field microscope and a fluorescence microscope to detect the decellularization efficiency. The results are as follows: Figure 2 As shown, compared with the undecellularized group, after treatment with cell lysis buffer, human astrocytes HA1800, human dermal fibroblasts (HDF), and human hair follicle stem cells (HFSC) all underwent cell lysis, with no clearly visible cells. DAPI staining revealed that after DNase I digestion, nucleic acids were fragmented, with no clearly visible cell nuclei, and this will not affect the subsequent application of extracellular matrix-coated cell culture plates.

[0065] Test Example 2 1. Test samples: The decellularized extracellular matrix obtained in step (7) of step 2 in Examples 1-3 and Matrigel (catalog number 356234) purchased from Corning were used as control samples.

[0066] 2. Immunofluorescence staining of collagen type I and fibronectin was performed on the test samples, followed by observation and photography under a fluorescence microscope. Results are as follows: Figure 3 As shown, Matrigel did not detect type I collagen and fibronectin, but human astrocyte extracellular matrix HA1800 ECM, human dermal fibroblast extracellular matrix HDF ECM, and human hair follicle stem cell extracellular matrix HFSC ECM were expressed to varying degrees.

[0067] 3. The sample to be tested was fixed and dehydrated, observed using a bright-field microscope, and detected using a scanning electron microscope. The results are as follows: Figure 4 As shown, different types of extracellular matrix exhibit distinct fibrous structures after fixation and dehydration. Under a scanning electron microscope, the fibrous network microstructure of human astrocyte extracellular matrix HA1800 ECM is even more obvious.

[0068] 4. The types and relative amounts of extracellular proteins in the samples were determined using 4D label-free quantitative proteomics analysis with a mass spectrometer. The results are shown in Table 1. The types of proteins in different types of extracellular matrix are basically the same, indicating that extracellular matrix proteins are relatively conserved in evolution. Figures 5-8 As shown, the relative content of the same protein varies in the extracellular matrix from different cell lines, suggesting that the expression of a specific protein differs across different cell lines.

[0069] Table 1. Statistical analysis of the number of different subclasses of common extracellular matrix proteins (species)

[0070] Test Example 3 1. Samples to be tested: Decellularized extracellular matrix obtained in step (7) of step 2 in Examples 1-3 (stored for 0 days), decellularized extracellular matrix obtained in step (8) of step 2 in Examples 1-3 (stored for 7 days), Matrigel stored for 0 days and Matrigel stored for 7 days.

[0071] 2. SDS-PAGE analysis was performed on the test samples to detect the stability of the extracellular matrix components. Results are as follows: Figure 9 As shown, after 7 days of storage at 4°C, Matrigel, human dermal fibroblast extracellular matrix HDF ECM, and human hair follicle stem cell extracellular matrix HFSC ECM will partially degrade, while human astrocyte extracellular matrix HA1800 ECM will hardly degrade.

[0072] 3. Using a 1% penicillin-streptomycin double antibiotic DPBS solution as a control, the test sample was spread onto antibiotic-free LB solid medium and incubated at 37°C for 24 h to detect the presence of bacteria in the extracellular matrix. Results are as follows: Figure 10 As shown, no colonies grew on the antibiotic-free LB solid medium, indicating that the extracellular matrix of human astrocyte extracellular matrix HA1800 ECM, human dermal fibroblast extracellular matrix HDF ECM, and human hair follicle stem cell extracellular matrix HFSC ECM can remain sterile after being stored for 7 days in DPBS solution containing 1% penicillin-streptomycin bispecific antibodies.

[0073] Example 4 The specific steps for inducing human dermal fibroblasts (HDF) to differentiate into neurons are as follows: (1) Human dermal fibroblasts were revived and expanded in 10 cm cell culture dishes.

[0074] (2) Digest the cells from step (1), discard the culture medium, add 3 mL of DPBS solution to wash 2-3 times, then add 1 mL of Trypsin-EDTA, shake to mix and cover the cells, and place in a 37℃ incubator for digestion.

[0075] (3) Add 3 mL of 10% FBS cell culture medium to stop digestion, mix the cells with a pipette, transfer the cell resuspension to a 15 mL EP tube, and centrifuge at 1000 rpm for 3 min at 4°C.

[0076] (4) Discard the supernatant, resuspend the cells in 10% FBS cell culture medium, count the cells, and seed 3.5 × 10⁻⁶ cells into each well of the extracellular matrix-coated cell culture plate (for direct use only, without storage treatment) obtained in step 2 of Example 1. 4 Cells were cultured in a 5% CO2, 37°C incubator.

[0077] (5) After 12 h, discard the original culture medium, wash 2-3 times with 0.5 mL DPBS solution, and replace with fresh culture medium.

[0078] (6) After 24 h, when the cell confluence reaches about 80%, add 10 mg / mL of polybrene at a ratio of 1:1000 to the knockdown cells. PTBP1 In the lentivirus, the final concentration of polybrene was adjusted to 0.01 mg / mL to obtain the first mixture. 0.5 mL of the first mixture was added to each well to infect human dermal fibroblasts (HDF).

[0079] (7) After 24 h, the first mixture was aspirated and 50 mg / mL hygromycin B was added to 2% FBS cell culture medium at a ratio of 1:500 to make the final concentration of hygromycin B 0.1 mg / mL, thus obtaining the second mixture. 0.5 mL of the second mixture was added to each well to screen for human dermal fibroblasts (HDF) that were not infected by lentivirus.

[0080] (8) After 24 hours, replace the second mixture once, and repeat once after 24 hours. Screen for 3 consecutive days.

[0081] (9) Discard the mixture, wash once with 0.5 mL DPBS solution, add 0.5 mL N3 medium to each well to induce human dermal fibroblasts (HDF) to differentiate into neurons, and change the N3 medium every 36 h. The N3 medium consists of 0.5 mL of penicillin-dextrin, 125 μL of 10 mg / mL insulin, 50 μL of 50 mg / mL transferrin, 50 μL of 20 μM progesterone, 50 μL of 100 μM putrescine, 50 μL of 30 μM sodium selenite and 49.5 mL DMEM / F12.

[0082] Comparative Example 1 Human dermal fibroblasts (HDF) were induced to differentiate in the manner described in Example 4, with the only difference being that step (4) used a Matrigel-coated cell culture plate.

[0083] Comparative Example 2 Human dermal fibroblasts (HDF) were induced to differentiate in the manner described in Example 4. The only difference was that in step (4), the cell culture plate was coated with the extracellular matrix obtained in step 2 of Example 2 (for direct use without storage).

[0084] Comparative Example 3 Human dermal fibroblasts (HDF) were induced to differentiate in the manner described in Example 4. The only difference was that in step (4), the cell culture plate was coated with the extracellular matrix obtained in step 2 of Example 3 (for direct use without storage).

[0085] Test Example 4 Biocompatibility testing In Example 4 and Comparative Examples 1-3, human dermal fibroblasts were seeded in extracellular matrix-coated cell culture plates for 24 h after HDF treatment. The relative viability of the cells was characterized using DAPI staining. Results are as follows: Figure 11 and Figure 12 As shown, human dermal fibroblasts (HDFs) can survive normally on four types of extracellular matrix-coated cell culture plates, confirming that extracellular matrix-coated cell culture plates do not have a toxic effect on cells. The relative viability of human dermal fibroblasts (HDFs) on the four types of extracellular matrix-coated cell culture plates showed essentially no difference, further confirming the feasibility of preparing extracellular matrix-coated cell culture plates.

[0086] Test Example 5 Samples were collected on days 9, 14, and 21 of step (9) in Examples 4 and Comparative Examples 1-3, and Tuj1 immunofluorescence staining was performed on decellularized human astrocyte extracellular matrix HA1800 ECM and the control group. The results are as follows: Figures 13-16As shown, in the HDF-induced transdifferentiation of human dermal fibroblasts into neurons on different types of extracellular matrix cell culture plates, on days 9, 14, and 21 of differentiation, the cell culture plates coated with human astrocyte extracellular matrix HA18000 ECM in Example 4 showed significant advantages for neuronal differentiation and survival, effectively improving the differentiation efficiency of neurons and promoting the survival of induced differentiated neurons.

[0087] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing cell-derived extracellular matrix, characterized in that, Includes the following steps: After obtaining gelatin-coated cell culture plates, the gelatin in the gelatin-coated cell culture plates is cross-linked with glutaraldehyde to obtain pretreated cell culture plates. Human astrocytes were cultured using the pretreated cell culture plate. When the cell confluence was ≥100%, the cells were cultured in a medium containing ascorbic acid to obtain the cultured cells. The cultured cells were lysed using a lysis buffer, and after cell removal, nucleic acid fragmentation was performed to obtain cell-derived extracellular matrix, which was then coated onto the cell culture plate. The cell lysis buffer consists of NH4OH, 20% v / v Triton X-100, and PBS, with a volume ratio of NH4OH, 20% v / v Triton X-100, and PBS of 1:1.25:47.

75.

2. The preparation method according to claim 1, characterized in that, The gelatin is a 0.2% gelatin solution, and the coating temperature is 37°C for 1 hour. The glutaraldehyde is a 1% glutaraldehyde solution, and the cross-linking time is 30 min; The volume ratio of the gelatin solution to the glutaraldehyde solution is 8:

5.

3. The preparation method according to claim 1, characterized in that, The cell culture plate is a 24-well cell culture plate, and the seeding density of human astrocytes is 7 × 10⁶. 4 One hole / hole.

4. The preparation method according to claim 1, characterized in that, The concentration of ascorbic acid in the culture medium containing ascorbic acid is 50 μg / mL, and the culture time using the culture medium containing ascorbic acid is 7 days.

5. The preparation method according to claim 1, characterized in that, The nucleic acid fragmentation was performed using DNase I solution.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The human astrocytes include human astrocytes HA1800.

7. Cell culture plates containing cell-derived extracellular matrix and / or coated with cell-derived extracellular matrix obtained by the preparation method according to any one of claims 1 to 6.

8. The use of the cell-derived extracellular matrix and / or cell culture plates coated with cell-derived extracellular matrix as described in claim 7 in one or more of the following: (1) Inducing human dermal fibroblasts to differentiate into neurons; (2) To prepare products for tissue repair and / or regeneration; (3) Organoid culture.

9. A method for inducing human dermal fibroblasts to differentiate into neurons, characterized in that, Includes the following steps: Human dermal fibroblasts were seeded using the cell-derived extracellular matrix as described in claim 7 and / or cell culture plates coated with the cell-derived extracellular matrix. When the cell confluence was ≥80%, lentivirus and polybrene were used to knock down the cells. PTBP1 Expression was selected using hygromycin B to obtain knockdown samples. PTBP1 Human dermal fibroblasts; Knock down PTBP1 Human dermal fibroblasts were induced to differentiate into neurons.

10. The method according to claim 9, characterized in that, The seeding density of the human dermal fibroblasts was 3.5 × 10⁻⁶. 4 One per hole; The concentration of the polyaluminum glycoside is 0.01 mg / mL; The concentration of hygromycin B is 0.1 mg / mL; The culture medium used for inducing differentiation includes N3 medium, the components of which include DMEM / F12, penicillin, insulin, transferrin, progesterone, putrescine, and sodium selenite.

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