Simple preparation of ECM paper scaffold and its application

Abstract

This invention belongs to the field of medical materials technology and discloses a simplified preparation method and application of an ECM paper scaffold. Specifically, an ECM paper scaffold is disclosed, prepared from decellularized ECM and hexafluoroisopropanol. The ECM paper scaffold provided by this invention has indentations and wrinkles of varying depths, as well as obvious through-holes, exhibiting good swelling rate, hydrophilicity, and permeability, and high tissue stability. Cells (such as HUVECs) can achieve rapid adhesion to the ECM paper scaffold, with visible tentacles adhering to the scaffold, and can extend and spread well, promoting cell proliferation, effectively promoting cell functional expression (such as promoting NO secretion by HUVECs), effectively promoting the deep penetration of nutrients and oxygen, and promoting cell growth.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to the simplified preparation and application of an ECM paper scaffold. Background Technology

[0002] Extracellular matrix (ECM), as a biomaterial, is widely used in tissue engineering due to its physiological functions and microstructure, as well as the presence of bioactive substances such as proteins and growth factors that promote cell growth, thereby facilitating cell migration, proliferation, differentiation, and functional expression. Currently used ECM biomaterials mainly include decellularized ECM derived from animal tissues and organs, as well as decellularized ECM derived from cell culture. However, applied research has revealed that decellularized ECM derived from animal tissues or organs suffers from batch-to-batch variability, complex composition, instability, strong immunogenicity, the risk of viral carriage, and limitations in source, significantly restricting its application in tissue engineering. In contrast, cell-derived decellularized ECM can achieve batch-to-batch, component-stable production through large-scale, controlled in vitro culture, while also exhibiting lower immunogenicity and avoiding the risk of viral carriage.

[0003] Stem cell therapy, as an effective treatment method in regenerative medicine, is a potential treatment option for widespread damage and degenerative diseases. Current research indicates that stem cell therapy has broad clinical application prospects. However, its therapeutic effects are still limited because current stem cell injection therapies cannot achieve prolonged accumulation of implanted cells at a specific implantation site, and require the implantation of a large number of cells to achieve the desired therapeutic effect. Therefore, due to these limitations, biomaterials are used as cell delivery carriers in stem cell therapy. Biomaterials promote cell attachment and proliferation, thereby delivering and accumulating cells at the implantation site for an extended period, and promoting cell migration, proliferation, differentiation, and functional expression. Biomaterials used as cell delivery carriers must possess excellent biocompatibility, mechanical properties, low immunogenicity, and the ability to maintain and promote cell migration and functional expression. Based on the excellent cell compatibility, low immunogenicity, and controllable mechanical properties of decellularized ECM, it can serve as an ideal biomaterial for cell delivery, promoting cell implantation, migration, proliferation, differentiation, and functional expression.

[0004] However, utilizing biomaterials as cell delivery carriers still faces certain challenges. For example, cells cultured on biomaterials grow on the material surface first and then gradually migrate into the material's interior. Achieving the ideal biomaterial-cell body requires a long construction period, thus hindering its application. Furthermore, in cell culture based on a biomaterial matrix, the material's microstructure, porosity, size, and thickness can all affect the penetration of oxygen and nutrients into the material, consequently impacting cell survival and growth. Moreover, a single biomaterial matrix cannot simulate the multi-layered physiological structure of biological tissues. Therefore, constructing an ideal biomaterial-cell body requires designing materials with appropriate dimensions and a reasonable microstructure and porosity to effectively promote oxygen and nutrient permeation, thus facilitating cell survival and growth. This, in turn, enables the construction of biomimetic multi-layered three-dimensional tissues, allowing for better adhesion to different damaged tissue sites and the implantation of cells to promote tissue repair and treatment. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the current situation and the prior art.

[0006] The first aspect of the present invention is to provide an ECM paper support.

[0007] The second objective of this invention is to provide a method for preparing the ECM paper scaffold of the first aspect of this invention.

[0008] The object of a third aspect of the present invention is to provide the application of the ECM paper scaffold of the first aspect of the present invention and / or the preparation method of the second aspect of the present invention in the preparation of tissue substitutes.

[0009] The fourth aspect of this invention aims to provide the application of the ECM paper scaffold of the first aspect of this invention and / or the preparation method of the second aspect of this invention in cell culture.

[0010] The fifth aspect of this invention aims to provide the application of the ECM paper scaffold of the first aspect of this invention and / or the preparation method of the second aspect of this invention in the preparation of tissue repair products.

[0011] The sixth aspect of this invention aims to provide a product.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides an ECM paper scaffold made from raw materials including decellularized ECM and hexafluoroisopropanol.

[0014] In some embodiments of the present invention, the mass-to-volume ratio of the decellularized ECM to hexafluoroisopropanol is (7-20):1.

[0015] In some embodiments of the present invention, the mass-to-volume ratio of the decellularized ECM to hexafluoroisopropanol is (7-15):1.

[0016] In some embodiments of the present invention, the mass-to-volume ratio of the decellularized ECM to hexafluoroisopropanol is (10-15):1.

[0017] In some embodiments of the present invention, the decellularized ECM includes cell-derived decellularized ECM or tissue-derived decellularized ECM.

[0018] In some embodiments of the present invention, the ECM paper is any one of different shapes such as circle, square or rectangle.

[0019] In some embodiments of the present invention, the size of the ECM paper is 0.787–2.362 mg / cm³. 2 .

[0020] A second aspect of the present invention provides a method for preparing an ECM paper scaffold according to the first aspect of the present invention, comprising the following steps: mixing decellularized ECM with hexafluoroisopropanol and solidifying it to obtain an ECM paper scaffold.

[0021] In some embodiments of the present invention, the mixing time is 15 to 40 hours.

[0022] In some embodiments of the present invention, the mixing time is 18 to 36 hours.

[0023] In some embodiments of the present invention, the mixing time is 20 to 30 hours.

[0024] In some embodiments of the present invention, the decellularized ECM is derived from any one of stem cells, fibroblasts, and tumor cells.

[0025] In some embodiments of the present invention, the method for preparing the decellularized ECM is to digest the cells, resuspend them in an adherent culture, and then differentiate them into primitive endoderm-like cells using a differentiation medium; the primitive endoderm-like cells are then decellularized by ammonia water to obtain the decellularized ECM.

[0026] In some embodiments of the present invention, the curing and molding process includes, but is not limited to, the following method: injecting a mixture of decellularized ECM and hexafluoroisopropanol into a mold, drying it, and then demolding it.

[0027] In some embodiments of the present invention, the mold is made of hydrophobic materials such as silicone or polytetrafluoroethylene.

[0028] In some embodiments of the present invention, the mold is composed of a substrate made of hydrophobic materials such as silicone or polytetrafluoroethylene and a hollow upper column.

[0029] In some embodiments of the present invention, the shape of the hollow upper column can be any of different shapes such as circle, square or rectangle.

[0030] In some embodiments of the present invention, the drying time is 2 to 6 hours.

[0031] A third aspect of the present invention provides the application of the ECM paper scaffold of the first aspect of the present invention and / or the preparation method of the second aspect of the present invention in any one of (1) to (4):

[0032] (1) Preparation of tissue substitutes;

[0033] (2) Drug screening;

[0034] (3) Case model studies;

[0035] (4) In vitro 3D tissue model.

[0036] A fourth aspect of the present invention provides the application of the ECM paper scaffold of the first aspect of the present invention and / or the preparation method of the second aspect of the present invention in any one of (5) to (8):

[0037] (5) Cell culture;

[0038] (6) Prepare products from cultured cells;

[0039] (7) Promotes the expression of cellular functions;

[0040] (8) Prepare products that promote cell function expression.

[0041] In some embodiments of the present invention, the cells include stem cells, fibroblasts, epithelial cells, myocytes, tumor cells, chondrocytes, osteocytes, glomerular mesangial cells, and endothelial cells, etc.

[0042] A fifth aspect of the present invention provides the application of the ECM paper scaffold of the first aspect of the present invention and / or the preparation method of the second aspect of the present invention in any one of (9) to (10):

[0043] (9) Tissue repair;

[0044] (10) Prepare tissue repair products.

[0045] A sixth aspect of the present invention is to provide a product comprising the ECM paper support of the first aspect of the present invention.

[0046] In some embodiments of the present invention, the product includes, but is not limited to, reagents, kits, and tissue substitutes.

[0047] In some embodiments of the present invention, the product is used for drug screening, cell culture, and promoting cell function expression.

[0048] The beneficial effects of this invention are:

[0049] The ECM paper scaffold provided by this invention has indentations and wrinkles of varying depths, as well as obvious through-holes, exhibiting good swelling rate, hydrophilicity, and permeability, and high tissue stability. Cells (such as HUVECs) can rapidly adhere to the ECM paper scaffold, with clearly visible tentacles adhering to it, and can extend and spread well, promoting cell proliferation, effectively promoting cell function expression (such as promoting NO secretion in HUVECs), effectively promoting the deep penetration and exudation of nutrients and oxygen, and promoting cell growth.

[0050] The ECM paper scaffold provided by this invention can serve as an ideal seed cell carrier for stem cell therapy research, and can also be used to construct multi-layered in vitro 3D tissue models for organoid-related research by combining cells and ECM paper scaffolds.

[0051] The method for preparing ECM paper scaffolds provided by this invention is simple to operate, has a short cycle time, and is low in cost, and can achieve quantitative and mass production (with stable ECM paper structure between batches). Attached Figure Description

[0052] Figure 1 The graph shows the detection results of the effects of different ECM concentrations and different dissolution times on the preparation of ECM paper.

[0053] Figure 2 This is a flowchart of the ECM paper preparation process and its macroscopic and microscopic structure; wherein, A is the specific process of ECM paper scaffold preparation, B is the prepared circular ECM paper scaffold, and C is the surface morphology of the prepared ECM paper scaffold.

[0054] Figure 3 The results of the test on the effect of different dissolution times on the formation of concave holes in ECM paper.

[0055] Figure 4 The images show the detection results of surface morphology and protein composition of different batches of ECM paper.

[0056] Figure 5 The test results are for the hydrophilicity and hydrophobicity of ECM paper scaffolds; where A is the swelling rate test of ECM paper scaffolds, B is the stability test of ECM paper scaffolds, and C is the contact angle test of ECM paper scaffolds.

[0057] Figure 6 The results show the cell adhesion and proliferation assays performed on the ECM paper scaffold; A represents the cell adhesion results, B represents the fluorescence staining results, and C represents the CCK8 assay results.

[0058] Figure 7 The image shows the detection results of how ECM paper scaffolds promote endothelial cell functional expression.

[0059] Figure 8 This is a graph showing the results of ECM paper permeability testing. Detailed Implementation

[0060] The present invention will now be described in detail with reference to specific embodiments, but this does not limit the scope of the invention.

[0061] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available.

[0062] The specific process of F9 cell differentiation culture and extraction of decellularized ECM is as follows:

[0063] (1) Resuscitate and expand mouse teratoma cells (F9 cells): Add F9 cells to complete culture medium (based on high glucose DMEM, with double antibiotics accounting for 1% of the culture medium volume and FBS accounting for 10% of the culture medium volume) and culture at 37℃ for 30h.

[0064] (2) Digest the cells from step (1) (add 1 mL of digestion solution (0.25% Trypsin-0.53 mM EDTA) and digest at room temperature for 1 min), centrifuge at 800 rpm for 5 min and discard the supernatant, add 5 mL of complete culture medium containing 10% serum to resuspend the cells and count them.

[0065] (3) The cells counted in step (2) are divided into groups of 4.5 × 10⁻⁶. 6 Re-inoculate in 10cm culture dishes, add complete culture medium, and incubate at 37℃ for 24 hours in an incubator.

[0066] (4) Replace the culture medium in step (3) with differentiation medium (based on DMEM (high glucose, 4 g / L) culture medium, wherein the double antibiotic accounts for 1% of the culture medium volume, FBS accounts for 10% of the culture medium volume, the concentration of all-trans retinoic acid is 0.1 μM and the concentration of bibutyryl-cyclic adenosine monophosphate is 250 μM), and culture F9 cells for differentiation for 3 days to form primitive endoderm-like cells;

[0067] (5) Wash the primitive endoderm-like cells obtained in step (4) twice with deionized water, add ammonia water with a concentration of 25mM, and place the cells on a decolorizing shaker at 4℃ for decellularization treatment for 1 hour.

[0068] (6) Place the cell sample after decellularization in step (5) into a dialysis bag (1000D) and stir in deionized water at room temperature (about 25°C) for 24 hours, changing the water 5 to 10 times during the process.

[0069] (7) The cell sample washed in step (6) was freeze-dried at -80°C to obtain decellularized extracellular matrix containing laminin and collagen IV, i.e., decellularized ECM.

[0070] Example 1

[0071] A method for preparing an ECM paper scaffold includes the following steps:

[0072] (1) F9 cells were differentiated and cultured and decellularized ECM was extracted;

[0073] (2) Add 20 mg of decellularized ECM differentiated from F9 in step (1) to 2 mL of hexafluoroisopropanol (HFIP, Macklin, H811026, 99.5%) solution;

[0074] (3) Stir the HFIP solution containing decellularized ECM from step (2) at room temperature (25°C, the same below) for 24 hours to promote ECM dissolution and obtain a solution;

[0075] (4) Take 400uL of the solution from step (3) and drop it into the ECM paper scaffold to prepare the model (hollow silicone cylinder with an inner diameter of 1.85cm), and let it air dry at room temperature for 3 hours.

[0076] (5) Remove the dried ECM paper support from the mold in step (4) to obtain the ECM paper support.

[0077] Example 2

[0078] A method for preparing an ECM paper scaffold includes the following steps:

[0079] (1) F9 cells were differentiated and cultured and decellularized ECM was extracted;

[0080] (2) Add 20 mg of decellularized ECM differentiated from F9 in step (1) to 2 mL of hexafluoroisopropanol (HFIP, Macklin, H811026, 99.5%) solution;

[0081] (3) Stir the HFIP solution containing decellularized ECM from step (2) at room temperature (25°C, the same below) for 30 h to promote ECM dissolution and obtain a solution;

[0082] (4) Take 400uL of the solution from step (3) and drop it into the ECM paper scaffold to prepare the model (hollow silicone cylinder with an inner diameter of 1.85cm), and let it air dry at room temperature for 3 hours.

[0083] (5) Remove the dried ECM paper support from the mold in step (4) to obtain the ECM paper support.

[0084] Example 3

[0085] A method for preparing an ECM paper scaffold includes the following steps:

[0086] (1) F9 cells were differentiated and cultured and decellularized ECM was extracted;

[0087] (2) Add 16 mg of decellularized ECM differentiated from F9 in step (1) to 2 mL of hexafluoroisopropanol (HFIP, Macklin, H811026, 99.5%) solution;

[0088] (3) Stir the HFIP solution containing decellularized ECM from step (2) at room temperature (25°C, the same below) for 24 hours to promote ECM dissolution and obtain a solution;

[0089] (4) Take 400uL of the solution from step (3) and drop it into the ECM paper scaffold to prepare the model (hollow silicone cylinder with an inner diameter of 1.85cm), and let it air dry at room temperature for 3 hours.

[0090] (5) Remove the dried ECM paper support from the mold in step (4) to obtain the ECM paper support.

[0091] Example 4

[0092] A method for preparing an ECM paper scaffold includes the following steps:

[0093] (1) F9 cells were differentiated and cultured and decellularized ECM was extracted;

[0094] (2) Add 30 mg of decellularized ECM differentiated from F9 in step (1) to 2 mL of hexafluoroisopropanol (HFIP, Macklin, H811026, 99.5%) solution;

[0095] (3) Stir the HFIP solution containing decellularized ECM from step (2) at room temperature (25°C, the same below) for 24 hours to promote ECM dissolution and obtain a solution;

[0096] (4) Take 400uL of the solution from step (3) and drop it into the ECM paper scaffold to prepare the model (hollow silicone cylinder with an inner diameter of 1.85cm), and let it air dry at room temperature for 3 hours.

[0097] (5) Remove the dried ECM paper support from the mold in step (4) to obtain the ECM paper support.

[0098] Example 5

[0099] A method for preparing an ECM paper scaffold includes the following steps:

[0100] (1) F9 cells were differentiated and cultured and decellularized ECM was extracted;

[0101] (2) Add 30 mg of decellularized ECM differentiated from F9 in step (1) to 2 mL of hexafluoroisopropanol (HFIP, Macklin, H811026, 99.5%) solution;

[0102] (3) Stir the HFIP solution containing decellularized ECM from step (2) at room temperature (25°C, the same below) for 30 h to promote ECM dissolution and obtain a solution;

[0103] (4) Take 400uL of the solution from step (3) and drop it into the ECM paper scaffold to prepare the model (hollow silicone cylinder with an inner diameter of 1.85cm), and let it air dry at room temperature for 3 hours.

[0104] (5) Remove the dried ECM paper support from the mold in step (4) to obtain the ECM paper support.

[0105] Example 6

[0106] A method for preparing an ECM paper scaffold includes the following steps:

[0107] (1) F9 cells were differentiated and cultured and decellularized ECM was extracted;

[0108] (2) Add 5 mg of decellularized ECM differentiated from F9 in step (1) to 2 mL of hexafluoroisopropanol (HFIP, Macklin, H811026, 99.5%) solution;

[0109] (3) Stir the HFIP solution containing decellularized ECM from step (2) at room temperature (25°C, the same below) for 24 hours to promote ECM dissolution and obtain a solution;

[0110] (4) Take 400uL of the solution from step (3) and drop it into the ECM paper scaffold to prepare the model (hollow silicone cylinder with an inner diameter of 1.85cm), and let it air dry at room temperature for 3 hours.

[0111] (5) Remove the dried ECM paper support from the mold in step (4) to obtain the ECM paper support.

[0112] Example 7

[0113] A method for preparing an ECM paper scaffold includes the following steps:

[0114] (1) F9 cells were differentiated and cultured and decellularized ECM was extracted;

[0115] (2) Add 10 mg of decellularized ECM differentiated from F9 in step (1) to 2 mL of hexafluoroisopropanol (HFIP, Macklin, H811026, 99.5%) solution;

[0116] (3) Stir the HFIP solution containing decellularized ECM from step (2) at room temperature for 24 hours to promote ECM dissolution and obtain a solution;

[0117] (4) Take 400uL of the solution from step (3) and drop it into the ECM paper scaffold to prepare the model (hollow silicone cylinder with an inner diameter of 1.85cm), and let it air dry at room temperature for 3 hours.

[0118] (5) Remove the dried ECM paper support from the mold in step (4) to obtain the ECM paper support.

[0119] Example 8

[0120] A method for preparing an ECM paper scaffold includes the following steps:

[0121] (1) F9 cells were differentiated and cultured and decellularized ECM was extracted;

[0122] (2) Add 20 mg of decellularized ECM differentiated from F9 in step (1) to 2 mL of hexafluoroisopropanol (HFIP, Macklin, H811026, 99.5%) solution;

[0123] (3) Stir the HFIP solution containing decellularized ECM from step (2) at room temperature for 6 hours to promote ECM dissolution and obtain a solution;

[0124] (4) Take 400uL of the solution from step (3) and drop it into the ECM paper scaffold to prepare the model (hollow silicone cylinder with an inner diameter of 1.85cm), and let it air dry at room temperature for 3 hours.

[0125] (5) Remove the dried ECM paper support from the mold in step (4) to obtain the ECM paper support.

[0126] Example 9

[0127] A method for preparing an ECM paper scaffold includes the following steps:

[0128] (1) F9 cells were differentiated and cultured and decellularized ECM was extracted;

[0129] (2) Add 20 mg of decellularized ECM differentiated from F9 in step (1) to 2 mL of hexafluoroisopropanol (HFIP, Macklin, H811026, 99.5%) solution;

[0130] (3) Stir the HFIP solution containing decellularized ECM from step (2) at room temperature (25°C, the same below) for 12 hours to promote ECM dissolution and obtain a solution;

[0131] (4) Take 400uL of the solution from step (3) and drop it into the ECM paper scaffold to prepare the model (hollow silicone cylinder with an inner diameter of 1.85cm), and let it air dry at room temperature for 3 hours.

[0132] (5) Remove the dried ECM paper support from the mold in step (4) to obtain the ECM paper support.

[0133] Effect Example

[0134] 1. The effect of ECM solution concentration on the preparation of ECM paper scaffolds

[0135] By comparing the ECM paper scaffolds of Examples 1, 6, and 7, it was found that... Figure 1It can be seen that the 2.5 mg / mL ECM solution (Example 6) could not successfully prepare ECM paper, the 5 mg / mL ECM solution (Example 7) could form a film, but could not prepare a complete ECM paper, while the 10 mg / mL ECM solution (Example 1) could successfully prepare a complete ECM paper. This shows that the concentration of the ECM solution can affect the successful preparation of ECM paper and is an influencing factor for the successful preparation of ECM paper.

[0136] 2. Effect of ECM dissolution time on the preparation of ECM paper scaffolds

[0137] By comparing the ECM paper scaffolds of Examples 1, 8, and 9, it was found that due to the different ECM dissolution times, while film formation was promoted when the ECM dissolution time was 6 hours (Example 8) and 12 hours (Example 9), the preparation of an intact and undamaged ECM paper could not be promoted. However, ECM dissolved for 24 hours (Example 1) was able to promote the preparation of an intact and undamaged ECM paper. Figure 1 The results indicate that different dissolution times can affect the successful preparation of ECM paper, and are a factor influencing the successful preparation of ECM paper.

[0138] 3. Morphology analysis of ECM paper scaffolds.

[0139] The ECM Paper scaffold prepared in Example 1 was photographed; the prepared ECM Paper scaffold was cut into a square, the square ECM paper was adhered to a stage with conductive adhesive, and placed in an ion sputtering instrument for vacuum gold sputtering for 60s. After sputtering, the prepared sample was placed in a scanning electron microscope for surface morphology observation and photographing.

[0140] The results are as follows Figure 2 As shown, the surface of the ECM paper scaffold prepared in Example 1 has wrinkles and pits of different depths, some of which can penetrate the entire ECM paper scaffold.

[0141] 4. Detection of concave holes using ECM paper support

[0142] The ECM paper supports prepared in Example 1, Comparative Examples 3 and 4 were cut into squares, sputtered with gold, and their surface pits were observed using a scanning electron microscope.

[0143] The results are as follows Figure 3As shown, ECM paper prepared from ECM solutions dissolved for 6 hours (Comparative Example 3) and 12 hours (Comparative Example 4) exhibits indentations and wrinkles of varying depths, but no obvious through-holes are observed. In contrast, ECM paper prepared from ECM solutions dissolved for 24 hours (Example 1) shows indentations and wrinkles of varying depths, as well as obvious through-holes, which are uniformly distributed on the ECM paper. This indicates that sufficient dissolution of ECM promotes the formation of holes in the ECM paper.

[0144] 5. Composition and morphology analysis of ECM papers prepared in different batches

[0145] Different batches of ECM paper scaffolds were prepared according to the preparation method in Example 1, and cut into squares. After gold sputtering, their surface morphology was observed using a scanning electron microscope. Simultaneously, the cut ECM paper scaffolds were subjected to immunofluorescence staining (the general steps are as follows: 1) ECM paper was washed twice with PBS; 2) 2% bovine serum albumin was used for blocking at 25°C for 30 min; 3) Collagen IV and Laminin protein primary antibodies were incubated at 25°C for 2 h; 4) PBS was washed four times, 5 min each time; 5) Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0423, 1:500) was incubated at 25°C for 1 h; 6) PBS was washed four times, 5 min each time; 7) Observation and photography were performed using a fluorescence microscope).

[0146] The results are as follows Figure 4 As shown, under the same preparation conditions, the ECM paper prepared in different batches has a similar surface structure, with wrinkles and pores of varying depths, some of which can penetrate the entire ECM paper scaffold. CollagenIV and Laminin fluorescent staining showed that the ECM paper prepared in different batches had similar intensity of fluorescence staining. These results indicate that under the same preparation conditions, the ECM paper structure is stable between batches, and the Collagen IV and Laminin protein components are preserved in greater quantities and are relatively stable, thus ensuring the stability of the ECM paper between batches.

[0147] 6. Swelling rate detection of ECM paper scaffolds

[0148] The swelling rate of the ECM paper scaffold prepared in Example 1 was tested, and the testing process is as follows:

[0149] Weigh the dry weight (m1) of the ECM paper scaffold and weigh the wet weight (m2) of the ECM paper scaffold after soaking in 1× sterile PBS (Gibco, CT10010500CP) for 1 h and 24 h respectively. Calculate the swelling rate of the ECM paper scaffold based on the weight of the ECM paper scaffold in both dry and wet states. The calculation formula is as follows: Swelling rate = [(m2-m1) / m1×100%].

[0150] The results showed that the swelling rate of the ECM paper scaffold reached 189% after 1 hour of immersion. After 24 hours of immersion, the swelling rate was not significantly different from that after 1 hour, remaining at 192.6%. Figure 5 (A) indicates that the ECM paper scaffold swells rapidly and remains relatively stable.

[0151] 7. ECM paper stent for stent stability assessment

[0152] The stability of the ECM paper scaffold prepared in Example 1 was evaluated as follows:

[0153] Weigh the dry weight (m1) of the ECM paper scaffold prepared in Example 1. Soak the weighed ECM paper scaffold in 1× sterile PBS and place it in a 37°C environment for 1 day, 3 days, 5 days, 7 days and 10 days. At the corresponding soaking time points, dry the soaked ECM paper scaffold again and weigh it (m2). Calculate the degradation rate of the ECM paper scaffold based on the weight of the ECM paper scaffold before and after soaking. The calculation formula is as follows: Degradation rate = [(m2-m1) / m1×100%].

[0154] Experimental results showed that the ECM paper scaffold gradually degraded in PBS after soaking for 1–7 days, and the degradation of the ECM paper scaffold reached a relatively stable level after day 10. Figure 5 (B) It should be noted that although the degradation rate of the ECM paper scaffold on day 10 was slightly higher than that on day 7, this is because there is a certain error in the detection, which is acceptable in the experiment, and statistical comparison showed that the difference was not statistically significant.

[0155] 8. Hydrophilicity testing of ECM paper scaffolds

[0156] The hydrophilicity of the ECM paper scaffold prepared in Example 1 was tested, as follows:

[0157] Cut the ECM paper holder into small squares and lay them flat on a glass slide. Place the slide on a contact angle meter to measure the size of the water droplet contact angle of the ECM paper holder.

[0158] The results showed that after the water droplet contacted the ECM paper support, the contact angle rapidly decreased to about 30° within 5 seconds, and further decreased to about 20° within 60 seconds. Figure 5 (C)

[0159] 9. Application of ECM paper scaffolds in cell culture

[0160] The ECM paper scaffold prepared in Example 1 was immersed in 75% alcohol for 30 min for disinfection; the alcohol was removed, and the ECM paper scaffold was washed twice with sterile 1×PBS; the washed ECM paper scaffold was placed under ultraviolet light for 30 min while moistened; then it was immersed in sterile 1×PBS at 4°C for 2 days; simultaneously, human umbilical vein endothelial cells (HUVECs) were resuscitated, expanded, and cultured, and then digested with 0.25% trypsin, centrifuged, resuspended, and counted; the HUVECs were then divided into 5×10 groups. 4 HUVECs were seeded onto ECM paper scaffolds (1.85 cm in diameter) and cultured in complete culture medium containing 10% serum. Simultaneously, HUVECs cultured in 24-well plates served as a control. Finally, the ECM paper scaffolds containing HUVECs were subjected to immunofluorescence, CCK-8 assay, and NO secretion detection.

[0161] Immunofluorescence: HUVECs adhered to the ECM paper scaffold and control cells for 24 hours were photographed under a microscope. The adhered cells were stained with the cytoskeletal protein F-actin and photographed. The steps were as follows: 1) Wash the HUVECs on the control and ECM paper with PBS three times; 2) Fix with 3.7% paraformaldehyde for 10 min; 3) Wash with PBS three times; 4) Disrupt cell membranes with 0.1% Triton X-100 PBS for 10 min; 5) Wash with PBS three times; 6) Stain with Actin-Tracker Red-Rhodamine (microfilament red fluorescent probe, Beyotime, C2207S) for 20 min; 7) Wash with PBS twice; 8) Stain with DAPI for 5 min; 9) Wash with PBS, observe and photograph under a fluorescence microscope.

[0162] CCK8: HUVECs cells were cultured on ECM paper scaffolds, and cell proliferation was assessed using the CCK8 kit (GLPBIO, GK10001) at days 1, 3, 5, 7, and 10. The procedure was as follows: First, the culture medium was aspirated, and the cells were washed twice with PBS. Fresh culture medium was added to each well (200 μL), along with 20 μL of CCK-8 reagent. After mixing, the cells were incubated in an incubator for 1 hour. Then, the culture medium was aspirated into 96-well plates, and the OD values ​​were measured and calculated using a microplate reader at 450 nm. The control group was also assessed using the same method.

[0163] NO secretion detection: Supernatant from HUVECs cultured on ECM paper scaffolds for 1, 3, 5, 7, 10, and 14 days was extracted, and NO secretion was detected using a NO detection kit (Beyotime, S0021S). The steps were as follows: 1) Collect 24-hour cell culture supernatant at the corresponding time points and centrifuge at 1000 rpm for 1 min; 2) Dilute the kit standards with serial concentrations; 3) Add 50 μL each of the standard and sample to a 96-well plate; 4) Add 50 μL of Griess I reagent to each well; 5) Add 50 μL of Griess II reagent to each well and mix well; 6) Measure the absorbance at 540 nm using a microplate reader; 7) Calculate the NO secretion concentration of the sample based on the standard sample. The control group was detected using the same method.

[0164] The results are as follows Figure 6 As shown, compared to adhesion on culture dishes, HUVECs can also achieve rapid adhesion on ECM paper scaffolds, and obvious tentacles are visible adhering to the ECM paper scaffolds. Figure 6 (As indicated by the black and red arrows in section A); Fluorescent staining results further showed that cells on the ECM paper scaffold had more tentacles, promoting cell adhesion to the ECM paper scaffold, and cells adhering to the ECM paper scaffold had a larger cell area morphology, indicating that cells could better extend and spread on the ECM paper scaffold. Figure 6 (B). CCK8 results showed that when cells were cultured on ECM paper scaffolds, cell proliferation gradually increased significantly within 1–7 days, and cell proliferation stabilized relatively after 7 days. Figure 6 The results showed that culture on ECM paper scaffolds promoted NO secretion in HUVECs at 1, 3, 10, and 14 days of culture, indicating that ECM paper scaffolds promote the functional expression of HUVECs. Figure 7 ).

[0165] 10. Permeability testing of ECM paper scaffolds

[0166] The permeability of the ECM paper scaffold prepared in Example 1 was tested, as follows:

[0167] Fix or double-layer the ECM paper scaffold prepared in Example 1 onto filter paper; drop 20 μL of PBS into the center of the ECM paper scaffold and observe the PBS penetration.

[0168] The results are as follows Figure 8 As shown, water droplets can be seen rapidly penetrating through a single-layer ECM paper and wetting the filter paper. Water droplets can also be seen penetrating through both layers of ECM paper and wetting the filter paper in a double-layered ECM paper. This indicates that the prepared ECM paper has excellent permeability and can effectively promote the deep penetration of nutrients and oxygen into the interior of cells when applied to 3D tissue construction, thereby promoting cell growth.

[0169] The ECM paper scaffolds prepared in Examples 2-5 are not significantly different from the ECM paper scaffold in Example 1 in terms of morphology and properties. Due to space limitations, they will not be described in detail.

[0170] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An ECM paper scaffold, prepared by the following method: decellularized ECM is mixed with hexafluoroisopropanol and solidified to obtain an ECM paper scaffold, wherein the ECM paper scaffold has indentations and folds of different depths and through-holes, wherein the mass-to-volume ratio of decellularized ECM to hexafluoroisopropanol is (8-15) mg:1 mL, and the mixing time is 24-30 h.

2. The ECM paper support according to claim 1, characterized in that, The decellularized ECM includes cell-derived decellularized ECM or tissue-derived decellularized ECM.

3. The ECM paper support according to any one of claims 1 to 2, characterized in that, The method for preparing decellularized ECM involves digesting and resuspending cells for adherent culture, then differentiating them into primitive endoderm-like cells using differentiation medium; the primitive endoderm-like cells are then decellularized using ammonia water to obtain decellularized ECM.

4. The application of the ECM paper support according to any one of claims 1 to 3 in any one of (1) to (4): (1) Preparation of tissue substitutes; (2) Drug screening; (3) Case model studies; (4) Construct an in vitro 3D tissue model.

5. The application of the ECM paper support according to any one of claims 1 to 3 in any one of (5) to (8): (5) Cell culture; (6) Prepare products from cultured cells; (7) Promoting cell function expression for non-therapeutic purposes; (8) Prepare products that promote cell function expression.

6. The use of the ECM paper scaffold according to any one of claims 1 to 3 in the preparation of tissue repair products.

7. A product comprising the ECM paper holder according to any one of claims 1 to 3.

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