Preparation method and application of cell-engineered human collagen and extracellular matrix thereof

Abstract

The invention belongs to the field of cell biology, and particularly relates to a preparation method and application of an extracellular matrix of cell-engineered human collagen. The preparation method comprises the following steps: S1, culturing human induced pluripotent stem cells or a cell culture comprising the human induced pluripotent stem cells, and directly inducing differentiation to obtain induced mesenchymal stem cells or a cell culture comprising the induced mesenchymal stem cells; and S2, carrying out 3D culture on the induced mesenchymal stem cells through a 3D porous scaffold carrier. The extracellular matrix prepared by the preparation method provided by the invention is rich in collagen, so that the mesenchymal stem cells can be better promoted to be differentiated into osteoblasts, and meanwhile, a new thought is provided for the preparation of the collagen.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cell biology, and particularly relates to a preparation method of a cell-engineered human collagen extracellular matrix and application thereof. BACKGROUND

[0002] The composition of extracellular matrix determines the function of extracellular matrix, and the complete structure of extracellular matrix provides physical support for normal physiological activities of tissue cells and provides a scaffold function for cell regeneration. The extracellular matrix biological scaffold has been applied to tissue regeneration and functional reconstruction, and has great significance for the repair and regeneration of damaged tissues and organs. The molecular composition of extracellular matrix is specifically combined with cells, other matrix molecules and soluble factors in time and space, which can regulate cell adhesion, proliferation and transfer, differentiation and interaction between cells, and regulate the biological function of cells to provide a suitable microenvironment for cells. In recent years, it has been found that extracellular matrix is involved in immune response in both homeostasis and pathological state. Extracellular matrix is related to aging of human body. With the increase of age, the skin regeneration ability and remodeling ability decrease, and the total amount of collagen and elastin in the dermis layer of skin also decreases, leading to qualitative degradation of tissue components. These processes lead to facial skin relaxation, wrinkles and surface changes, including rough and dry skin diseases. Different extracellular matrix components have different functions. For example, collagen has the functions of beauty (anti-wrinkle, moisturizing, whitening, weight loss, breast augmentation) in addition to the function of beauty. Studies have shown that collagen also has the effects of preventing osteoporosis, improving joint health, improving blood circulation, improving stomach, improving human immunity, etc. Fibronectin can promote wound repair and healing (such as burns, trauma, etc.), and has the advantages of no infection, no scar and no side effects. At present, there is a problem of insufficient source of extracellular matrix.

[0003] Human pluripotent stem cells include human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPSC), which can be proliferated in vitro indefinitely and maintain their potential to differentiate into almost all adult cells. Induced mesenchymal stem cells (iMSC) obtained by directional induction and differentiation of human pluripotent stem cells can provide a stable seed cell for mesenchymal stem cell cell therapy. Mesenchymal stem cells (MSCs) are a kind of stem cells that can self-renew and have multi-directional differentiation potential and immune regulation function. Mesenchymal stem cells can differentiate into fibroblasts, osteoblasts, chondroblasts and other cells, which are the main cell types for synthesizing and secreting collagen. Collagen also has a regulatory effect on the behavior of mesenchymal stem cells. Different types of collagen can affect the adhesion, proliferation and differentiation of mesenchymal stem cells. For example, type I collagen can promote the differentiation of mesenchymal stem cells into osteoblasts.

[0004] Mesenchymal stem cells derived from human induced pluripotent stem cells have many special advantages, such as avoiding the pain of patients in the acquisition process, faster self-renewal speed, lower immunogenicity, and fewer ethical issues. Chemical methods detect that the extracellular matrix derived from induced mesenchymal stem cells is rich in chemical components such as laminin, fibronectin and collagen. The extracellular matrix derived from induced mesenchymal stem cells is more active, promotes cell secretion more, and secretes more extracellular matrix components. Promoting induced mesenchymal stem cells to secrete more extracellular matrix can solve the problems of insufficient extracellular matrix source and immune rejection, amplify the functions of each other, and promote the application of mesenchymal stem cells and extracellular matrix. SUMMARY

[0005] The purpose of the present application is to provide a method for obtaining a cell-engineered human collagen extracellular matrix by differentiating human induced pluripotent stem cells (iPSC) into mesenchymal stem cells (iMSC) and culturing them in a 3D porous scaffold carrier.

[0006] According to a first aspect of the present application, the present application provides a method for preparing a cell-engineered human collagen extracellular matrix, comprising the following steps:

[0007] S1: culturing human induced pluripotent stem cells or cell cultures containing human induced pluripotent stem cells, directly inducing differentiation to obtain induced mesenchymal stem cells or cell cultures containing induced mesenchymal stem cells;

[0008] S2: 3D culture of the induced mesenchymal stem cells by a 3D porous scaffold carrier;

[0009] In step S2, the 3D culture comprises: dropping the induced mesenchymal stem cell suspension onto the 3D porous scaffold carrier, immersing the 3D porous scaffold carrier in the induced mesenchymal stem cell suspension; and sequentially using the mesenchymal stem cell expansion medium and the expression medium for 3D culture of the cells; the expression medium comprises the following components: a basic medium and an additive; the additive comprises the following components: GlutaMax additive, NEAA non-essential amino acid, ITS-X supplement, human blood albumin, EGF, bFGF, TGF-β and PDGF-BB.

[0010] Specifically, the 3D porous scaffold carrier used in the present application is a polyether sulfone 3D porous sponge-like scaffold carrier. Specifically, the 3D porous scaffold carrier is prepared by using polyether sulfone as the main raw material; and the 3D porous scaffold carrier is prepared by the following method: adding polyether sulfone into dimethyl sulfoxide for stirring and dissolving to obtain a polyether sulfone scaffold solution; mixing the polyether sulfone scaffold solution with sodium chloride particles to obtain a polyether sulfone / sodium chloride mixed solution; pouring the polyether sulfone / sodium chloride mixed solution into a mold, freezing, and then placing it in an ice-water mixture for phase separation soaking and precipitation to obtain a crystal-containing scaffold; and placing the crystal-containing scaffold in boiling water to remove sodium chloride to obtain a polyether sulfone 3D porous sponge-like scaffold.

[0011] Specifically, the 3D porous scaffold carrier is prepared by the following method: dissolving polyether sulfone and / or polysulfone in a good solvent to obtain a scaffold solution with a mass fraction of 5-30 wt%; mixing the scaffold solution with water-soluble crystalline particles at a mass ratio of 1:3-1:40 to obtain a mixed solution; scaffold molding: shaping the mixed solution to obtain a crystal-containing scaffold; and placing the crystal-containing scaffold in a dissolving solution to remove the crystals to obtain the sponge-like scaffold carrier. Using the above 3D porous scaffold as the carrier for 3D culture of the extracellular matrix, since it has a large specific surface area, good biocompatibility and good mechanical properties, it can provide the induced mesenchymal stem cells with the space required for cell growth and the nutrition exchange channel, and realize the growth and reproduction of the cells in three-dimensional space.

[0012] The present application finds that, in the scheme of 3D culture of induced mesenchymal stem cells (iMSC) obtained by inducing differentiation of human induced pluripotent stem cells (iPSC) to obtain extracellular matrix of cell engineered human collagen, the expression level of collagen in the extracellular matrix is significantly affected by using different expression culture media for 3D culture. The extracellular matrix obtained by selecting the expression culture medium containing the above components has a high expression level of collagen, that is, the extracellular matrix prepared by the above preparation method is rich in cell engineered human collagen.

[0013] In some embodiments of the present application, the extracellular matrix prepared by the preparation method provided by the present application is rich in cell engineered human collagen. Preferably, the cell engineered human collagen is type I collagen.

[0014] In some embodiments of the present application, the volume concentration of the GlutaMax additive is 0.5-2%; the volume concentration of the NEAA non-essential amino acid is 0.5-2%; the volume concentration of the ITS-X supplement is 0.5-2%; the volume concentration of the human blood albumin is 0.5-2%; the concentration of the EGF is 1-10 ng / ml; the concentration of the bFGF is 1-10 ng / ml; the concentration of the TGF-β is 1-10 ng / ml; and the concentration of the PDGF-BB is 1-10 ng / ml.

[0015] In particular, the volume concentration of the GlutaMax additive can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% or a range between any two of the aforementioned values. In particular, the volume concentration of the NEAA non-essential amino acid can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% or a range between any two of the aforementioned values. In particular, the volume concentration of the ITS-X supplement can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% or a range between any two of the aforementioned values. In particular, the volume concentration of the human blood albumin can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% or a range between any two of the aforementioned values. In particular, the concentration of the EGF can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml or a range between any two of the aforementioned values. In particular, the concentration of the bFGF can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml or a range between any two of the aforementioned values. In particular, the concentration of the TGF-β can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml or a range between any two of the aforementioned values. In particular, the concentration of the PDGF-BB can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml or a range between any two of the aforementioned values.

[0016] In some embodiments of the application, the basal medium is selected from any one or more of high glucose DMEM, Alpha-MEM, DMEM / F12 medium.

[0017] In some embodiments of the present application, the volume concentration of the GlutaMax additive is 1%; the volume concentration of the NEAA non-essential amino acid is 1%; the volume concentration of the ITS-X supplement is 1%; the volume concentration of the human blood albumin is 1%; the concentration of the EGF is 5 ng / ml; the concentration of the bFGF is 5 ng / ml; the concentration of the TGF-β is 5 ng / ml; and the concentration of the PDGF-BB is 8 ng / ml.

[0018] In some embodiments of the present application, in step S2, the 3D culture comprises: dropwise adding the induced mesenchymal stem cell suspension to the sterilized 3D porous scaffold carrier, so that the 3D porous scaffold carrier is immersed in the induced mesenchymal stem cell suspension, to obtain a mixed system of the scaffold and the cell suspension, adding the mesenchymal stem cell expansion medium to the mixed system, and performing secondary 3D culture; after the secondary 3D culture, replacing the mesenchymal stem cell expansion medium with the expression medium, and performing tertiary 3D culture.

[0019] In some embodiments of the present application, the time of the secondary 3D culture is 10-20 days; and the time of the tertiary 3D culture is 10-20 days. Specifically, the time of the secondary 3D culture can be 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or a range consisting of any two of the above-mentioned values. Specifically, the time of the tertiary 3D culture can be 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or a range consisting of any two of the above-mentioned values.

[0020] In some embodiments of the present application, the culture conditions of the 3D culture are that the temperature is 35-40℃, and the CO2 concentration is 2-6%. Specifically, the temperature of the 3D culture can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, or a range consisting of any two of the above-mentioned values. The CO2 concentration of the 3D culture can be 2%, 3%, 4%, 5%, 6%, or a range consisting of any two of the above-mentioned values.

[0021] In some embodiments of the present application, the medium is replaced every 3-5 days during the secondary 3D culture and the tertiary 3D culture.

[0022] In some embodiments of the present application, in step S2, the 3D culture comprises:

[0023] The sterilized 3D porous scaffold carrier is clamped into a rotating bottle, and the induced mesenchymal stem cell suspension is dropwise added to the sterilized 3D porous scaffold carrier, so that the 3D porous scaffold carrier is immersed in the induced mesenchymal stem cell suspension;

[0024] placing the flask in a culture box for primary 3D culture;

[0025] adding mesenchymal stem cell expansion medium to the flask after primary 3D culture, and placing the flask in a flask culture machine for secondary 3D culture;

[0026] after secondary 3D culture, replacing the mesenchymal stem cell expansion medium with expression medium, and placing the flask in a flask culture machine for tertiary 3D culture.

[0027] In some embodiments of the present application, the time for primary 3D culture is 1-5h. Specifically, the time for primary 3D culture can be 1h, 2h, 3h, 4h, 5h, or a range consisting of any two of the above values.

[0028] In some embodiments of the present application, the time for secondary 3D culture is 10-20 days. Specifically, the time for secondary 3D culture can be 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or a range consisting of any two of the above values.

[0029] In some embodiments of the present application, the time for tertiary 3D culture is 10-20 days. Specifically, the time for tertiary 3D culture can be 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or a range consisting of any two of the above values.

[0030] In some embodiments of the present application, the conditions for 3D culture are temperature of 35-40℃ and CO2 concentration of 2-6%. Specifically, the temperature for 3D culture can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, or a range consisting of any two of the above values. The CO2 concentration for 3D culture can be 2%, 3%, 4%, 5%, 6%, or a range consisting of any two of the above values.

[0031] In some embodiments of the present application, the medium is replaced every 3-5 days during secondary 3D culture and tertiary 3D culture.

[0032] In some embodiments of the present application, 30-70 pieces of 3D porous scaffold carriers are placed in a single flask. Specifically, based on the 3D porous scaffold carriers provided in the present application, 30-100 million cells can be plated on each carrier. Specifically, after placing the carriers in the flask, the flask is shaken slightly to reduce the gap between the carriers.

[0033] In some embodiments of the present application, the density of the induced mesenchymal stem cell suspension is (2.0-3.0) x 105 cells / ml; the used induced mesenchymal stem cells are P4-P12 generation induced mesenchymal stem cells. Specifically, the density of the induced mesenchymal stem cell suspension can be 2.0x10 5 cells / ml, 2.1x10 5 cells / ml, 2.2x10 5 cells / ml, 2.3x10 5 cells / ml, 2.4x10 5 cells / ml, 2.5x10 5 cells / ml, 2.6x10 5 cells / ml, 2.7x10 5 cells / ml, 2.8x10 5 cells / ml, 2.9x10 5 cells / ml, 3.0x10 5 cells / ml, or a range composed of any two of the above. Specifically, the used induced mesenchymal stem cells can be any one of P4, P5, P6, P7, P8, P9, P10, P11, P12 generation induced mesenchymal stem cells.

[0034] In some embodiments of the present application, the mesenchymal stem cell expansion culture medium comprises: a mesenchymal stem cell basic culture medium and an additive, the additive comprises the following components: human platelet lysate, ascorbic acid, GlutaMax additive, NEAA non-essential amino acid, ITS-X supplement, IGF, bFGF, PDGF-BB.

[0035] In some embodiments of the present application, the additive comprises the following components: 1-10% human platelet lysate, 30-70mM ascorbic acid, 0.5%-2% GlutaMax additive, 0.5%-2% NEAA non-essential amino acid, 0.5%-2% ITS-X supplement, 1-10ng / ml IGF, 1-10ng / ml bFGF, 1-10ng / ml PDGF-BB.

[0036] In particular, the amount of human platelet lysate in the mesenchymal stem cell expansion medium can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a range between any two of the aforementioned values, in terms of volume concentration. In particular, the amount of ascorbic acid can be 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM or a range between any two of the aforementioned values. In particular, the amount of GlutaMax additive can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% or a range between any two of the aforementioned values, in terms of volume concentration. In particular, the amount of NEAA non-essential amino acids can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% or a range between any two of the aforementioned values, in terms of volume concentration. In particular, the amount of ITS-X supplement can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% or a range between any two of the aforementioned values, in terms of volume concentration. In particular, the amount of IGF can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml or a range between any two of the aforementioned values. In particular, the amount of bFGF can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml or a range between any two of the aforementioned values. In particular, the amount of PDGF-BB can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml or a range between any two of the aforementioned values.

[0037] In some embodiments of the present application, the mesenchymal stem cell basal medium is selected from at least one of high-sugar DMEM, Alpha-MEM, and DMEM / F12 medium.

[0038] In some embodiments of the present application, the preparation method further comprises:

[0039] S3: subjecting the extracellular matrix culture of the cell-engineered human collagen obtained in step S2 to decellularization treatment;

[0040] S4: extracting the extracellular matrix rich in the cell-engineered human collagen.

[0041] In some embodiments of the present application, the decellularization treatment comprises:

[0042] The extracellular matrix culture is taken, the culture medium is removed, and the extracellular matrix culture is washed with water for injection;

[0043] The decellularization liquid is added to the washed extracellular matrix culture, and the decellularization treatment is performed, and after the decellularization treatment, the extracellular matrix culture is washed and then freeze-dried.

[0044] In some embodiments of the present application, the decellularization liquid comprises the following components: SDS with a mass concentration of 0.1%-1% and Triton X-100 with a mass concentration of 0.5%-2%.

[0045] In some embodiments of the present application, the extraction comprises:

[0046] The DMSO-acetic acid solution is added to the freeze-dried extracellular matrix culture, so that the carrier is dissolved in the solution, and centrifugation is performed; the DMSO-acetic acid solution is continuously added to the precipitate obtained after centrifugation, and centrifugation is continuously performed, and the process is repeated until the supernatant after centrifugation is added to water for injection, and no white flocculent precipitate is generated;

[0047] The final obtained precipitate is desalted, homogenized, and freeze-dried to obtain the extracellular matrix of the cell-engineered human collagen.

[0048] In some embodiments of the present application, the centrifugation is performed at 6000-10000 rpm for 5-15 min. In some embodiments of the present application, the desalting is performed until the conductivity of the supernatant is reduced to below 15 us / cm.

[0049] In some embodiments of the present application, the homogenization conditions are 800-1500 pa, and the number of homogenization is 2-4 times.

[0050] According to the second aspect of the present application, the present application further provides a cell-engineered human collagen extracellular matrix prepared by the preparation method according to any one of the first aspect of the present application.

[0051] Specifically, in some embodiments of the present application, the extracellular matrix is rich in cell-engineered human collagen.

[0052] In some embodiments of the present application, the cell-engineered human collagen is collagen type I.

[0053] According to the third aspect of the present application, the present application further provides a use of the extracellular matrix according to the second aspect of the present application in the field of biomaterials.

[0054] In some embodiments of the present application, the use includes a use in the field of biomedical materials, cosmetic skin care products, etc. Specifically, the biomedical materials include a use in the field of tissue engineering scaffolds, wound dressings, hemostatic materials, etc. The cosmetic skin care products include a use in the field of skin care products, cosmetic injections, etc.

[0055] For the use in the tissue engineering scaffold, collagen type I is the main component of human connective tissue, has good biocompatibility and biological activity, and sodium hyaluronate has excellent moisturizing and lubricating properties, which can provide a good living microenvironment for cells. The combination of the two can prepare a tissue engineering scaffold for repairing and replacing damaged tissues such as skin, cartilage, bone, etc. For the use in the wound dressing, collagen type I can promote wound healing and reduce scar formation, and sodium hyaluronate can keep the wound moist, prevent infection, and promote cell migration and proliferation. The wound dressing prepared from the two can provide an ideal environment for wound healing and accelerate the repair process of the wound. For the use in the hemostatic material, the material prepared from the combination of collagen type I and sodium hyaluronate has good hemostatic properties and can be used for surgical wound hemostasis, traumatic hemorrhage, etc. They can promote the aggregation of platelets and the activation of coagulation factors, accelerate blood coagulation, and thus achieve the purpose of hemostasis.

[0056] For the application in the field of skin care, type I collagen can increase the elasticity and firmness of the skin, reduce the production of wrinkles, and sodium hyaluronate can absorb a large amount of water to keep the skin moist and full. Adding them to skin care products such as emulsions, creams, serums, etc. can play the roles of moisturizing, anti-wrinkling, repairing skin barrier, etc.; for the application in the field of beauty injection, type I collagen and sodium hyaluronate are often used in the preparation of beauty injections, such as water-light needles, collagen injections, etc. These components are directly delivered to the dermis layer of the skin through injection, which can quickly improve the texture and appearance of the skin, increase the gloss and elasticity of the skin, and achieve the effect of beauty and skin care.

[0057] Compared with the prior art, the present application has the following beneficial effects:

[0058] (1) The present application provides a preparation method of cell-engineered human collagen extracellular matrix. In the preparation method provided by the present application, the expression culture medium is further improved during the 3D culture of induced mesenchymal stem cells (iMSC) obtained by inducing differentiation of human induced pluripotent stem cells (iPSC), so that the collagen protein in the extracellular matrix obtained by the 3D culture method of the present application has a high level of expression. That is, the extracellular matrix prepared by the preparation method provided by the present application contains abundant cell-engineered human collagen, i.e. type I collagen, which can be combined with sodium hyaluronate and other substances to prepare biomedical materials and beauty and skin care products, and also provides a new idea for the preparation of collagen protein.

[0059] (2) The preparation method of the present application can realize large-scale production of extracellular matrix. This preparation method not only makes the yield of extracellular matrix considerable, but also has excellent quality, which can stably meet the increasing demand for extracellular matrix in the field of tissue engineering and regenerative medicine, and provides a solid material basis for related research and clinical application.

[0060] (3) The extracellular matrix of cell-engineered human collagen prepared by the preparation method of the present application can be applied to various tissue repair and regeneration scenarios, such as skin wound repair, cartilage tissue engineering, bone defect repair, etc., providing a new solution for the treatment of related diseases. At the same time, this technical system also provides a reference for the culture of other cell types and the production of extracellular matrix. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 It is a display figure of the pluripotent cell morphology of iPSC cells in Example 1 of the present application;

[0062] Figure 2 It is a display figure of the morphology of iPSC-derived iMSC cells obtained in Example 2 of the present application;

[0063] Figure 3AThe proportion of CD90, CD73, CD105, CD45, and CD14, CD34, CD19, and HLA-DR phenotypes of iMSCs detected by flow cytometry in the effect example of the present application + + + -

[0064] Figure 3B The proportion of CD14, CD34, CD19, and HLA-DR phenotypes of iMSCs detected by flow cytometry in the effect example of the present application - - - - -

[0065] Figure 4 The detection results of the osteogenic and adipogenic differentiation abilities of iMSCs in the effect example of the present application; wherein,(A) is the detection result of the osteogenic differentiation ability of iMSCs,(B) is the detection result of the adipogenic differentiation ability of iMSCs. Figure 4 Figure 4

[0066] Figure 5 The amount of cells at different days of DAPI staining when iMSCs were cultured in 3D on a 3D porous scaffold carrier in the effect example of the present application;

[0067] Figure 6 The expression level of collagen I of the extracellular matrix extracted from iMSCs cultured in 3D on a carrier in the effect example of the present application;

[0068] Figure 7 The expression level of collagen I of the extracellular matrix obtained from iMSCs cultured in 3D on a carrier under different group modes in the effect example of the present application; wherein, 1, 5, and 9 are initial samples, 2, 6, and 10 are loading samples, 3, 7, and 11 are flow-through and rinsing samples, and 4, 8, and 12 are elution samples.

[0069] Figure 8 The expression level of collagen I of the extracellular matrix obtained from iMSCs cultured in 3D on a carrier based on adipose-derived mesenchymal stem cells (ADSCs) in the effect example of the present application; wherein, 9 is a loading sample, 10 is a flow-through and rinsing sample, and 11 is an elution sample. DETAILED DESCRIPTION

[0070] The technical solutions of the present application are further illustrated by specific examples below, and the specific examples do not represent a limitation on the protection scope of the present application. Some non-essential modifications and adjustments made by others according to the concept of the present application still fall within the protection scope of the present application. ​​​​​​​​​

[0071] The raw material components or reagents used in the embodiments of the present application are commercially available unless otherwise specified.

[0072] Example 1

[0073] The present embodiment provides a method for directly inducing human induced pluripotent stem cell-derived cells into mesenchymal stem cells, and the specific steps are as follows:

[0074] (I) Culture and passage of human induced pluripotent stem cells (iPSCs)

[0075] Human induced pluripotent stem cells (iPSCs) were used as initial cells, and the specific steps are as shown in Figure 1 According to the results shown in Figure 1 The iPSC cells were typically clonal colony growth characteristics, with clear clonal edges, tight cell contact within the colony, high nuclear-cytoplasmic ratio, uniform morphology, and no differentiated cells. The culture and passage of human induced pluripotent stem cells (iPSCs) are as follows:

[0076] Laminin-521 coated culture plates: Take Laminin-521 stock solution, dilute Laminin-521 to a final concentration of 5 μg / mL with DPBS (containing calcium and magnesium); take the diluted Laminin-521 and add it to the culture plate, and place it in the 2-8°C refrigerator overnight or at 37°C for at least 2h for coating. After the coating is completed, take it out from the refrigerator and place it at room temperature for 30min before use.

[0077] Vitronectin coated culture plates: Take Vitronectin stock solution, dilute Vitronectin stock solution to a final concentration of 10 μg / mL with DMEM / F12; take the diluted Vitronectin and add it to the culture plate, and place it in the 2-8°C refrigerator overnight or at room temperature for at least 1h for coating. After the coating is completed, take it out from the refrigerator and place it at room temperature for 30min before use.

[0078] Cell recovery: Transfer the thawed iPSC cell suspension to a new 15mL centrifuge tube, and gently add the balanced room temperature human induced pluripotent stem cell complete medium to the cell suspension while gently shaking the centrifuge tube; transfer the 15mL centrifuge tube containing the iPSC cell suspension into a low-speed refrigerated centrifuge, and centrifuge at a centrifugal force of 300g at room temperature for 3min; after centrifugation, discard the supernatant, add 10 μM Y27632-containing human induced pluripotent stem cell complete medium (mTeSR Plus) to resuspend the cells, and gently blow them to a volume ratio of 1:15 to inoculate into a preheated 12Well cell culture plate, and culture in a 37°C, 5% CO2 concentration, 95% humidity incubator. Change the liquid every 20-24h, and start cell passage when the cell coverage reaches more than 80%.

[0079] Cell passage: when the cell coverage reaches more than 80%, the cell culture plate is taken out of the incubator, the cells are washed once with DPBS (without calcium and magnesium), then the Versene digestive enzyme is added to the cell culture plate to digest the cells for 3-5 min, the cell colony dissociation state is observed under a microscope, and after the cells are completely dissociated, the human induced pluripotent stem cell complete culture medium (mTeSR Plus) is added to gently blow the cell suspension; another blank culture plate preheated to 37°C is taken out of the incubator, the cell suspension is added at a volume ratio of 1:20, and placed in a temperature-controlled incubator at 37°C, CO 2 The concentration is 5%, and the cell culture is cultured overnight. Every 20-24 hours, the cell medium is replaced, and when the cell coverage reaches more than 80%, the next cell passage is started. The cells are passaged to P20 according to the above method, i.e. P20 generation iPSC cells are obtained.

[0080] (II) Culture of iPSC

[0081] The P20 generation iPSC cells prepared in step (I) are normally cultured in the maintenance medium, and the maintenance medium used is mTeSR Plus. When the iPSC cells are cultured to a confluence of 80-90%, the iPSC cells are digested into a complete single cell suspension using Tryple digestive enzyme, resuspended in iPSC maintenance medium, and Rock inhibitor is added to the iPSC maintenance medium. After 24 hours of Rock inhibitor maintenance, the complete iPSC maintenance medium is replaced. The iPSC culture is cultured using a 12-well cell culture plate, the iPSC maintenance medium used is mTeSR Plus, the Rock inhibitor used is Y-27632, the concentration of the Rock inhibitor is 10 μM, and the confluence of the cultured iPSC cells is 30%-50%.

[0082] (III) Induced differentiation of iPSC cells into mesenchymal stem cells (iMSC)

[0083] The maintenance medium of the iPSC cells in step (II) is replaced with mesenchymal stem cell induction medium, and placed in a temperature-controlled incubator at 37°C, CO 2 The concentration is 5%, and the cell culture is cultured overnight. Every 20-24 hours, the cell medium is replaced, and when the cell coverage reaches more than 80%, the next cell passage is started. The cells are passaged to P20 according to the above method, i.e. P20 generation iPSC cells are obtained. 4 cells / cm 2cells / cm2, and placed in a 37℃ incubator with 5% CO2. 2 The cells were cultured in a 37℃ incubator with 5% CO2, and the cells were recorded as P1. The cells were subcultured to P3 according to the above steps, and P3 iMSC cells were obtained. The mesenchymal stem cell induction culture medium was prepared according to the following formula: mesenchymal stem cell serum-free basal medium (the basal medium was selected from DMEM / F12 medium), 5% human platelet lysate, 50 mM ascorbic acid, 1% GlutaMax additive, 1% NEAA non-essential amino acid, 1% ITS-X supplement, 5 ng / ml IGF, 5 ng / ml bFGF, 5 ng / ml PDGF-BB, 5 μM TGF-β inhibitor (A8301), 5 μM GSK3β inhibitor (CHIR99021), and 8 μM Rock inhibitor (Y-27632).

[0084] (4) iMSC expansion subculture

[0085] When the P3 iMSC cells obtained in step (3) reached about 80%-90% confluence, the cells were resuspended in 1 mL of mesenchymal stem cell expansion medium, and the cells were seeded at a cell density of 0.9 x 10 4 cells / cm2, and placed in a 37℃ incubator with 5% CO2. 2 The cells were cultured in a 37℃ incubator with 5% CO2, and the cells were recorded as P1. The cells were subcultured to P3 according to the above steps, and P3 iMSC cells were obtained. The mesenchymal stem cell induction culture medium was prepared according to the following formula: mesenchymal stem cell serum-free basal medium (the basal medium was selected from DMEM / F12 medium), 5% human platelet lysate, 50 mM ascorbic acid, 1% GlutaMax additive, 1% NEAA non-essential amino acid, 1% ITS-X supplement, 5 ng / ml IGF, 5 ng / ml bFGF, 5 ng / ml PDGF-BB, 5 μM TGF-β inhibitor (A8301), 5 μM GSK3β inhibitor (CHIR99021), and 8 μM Rock inhibitor (Y-27632).

[0086] Example 1

[0087] (1) iMSC cell morphology

[0088] The morphology of the P3 and subsequent generations of iMSC cells prepared according to Example 1 was observed, and the results are shown in FIG. 1. Figure 2As shown, P3-P12 generation cells can present parallel spiral MSC morphological characteristics, and no aging phenomena such as cell volume increase or proliferation slowing down are observed, and can stably proliferate and pass.

[0089] (2) Flow detection

[0090] The P8 generation iMSC cells prepared by Example 1 were collected for flow detection, and the detection results are shown in Figure 3. The corresponding iMSC cells have more than 95% expression of CD73 + , CD90 + and CD105 + , which are positive, and less than 2% expression of CD19 - , CD14 - , CD34 - , CD45 - and HLA-DR - , which are negative.

[0091] (3) Detection of osteogenic and adipogenic differentiation ability

[0092] The osteogenic and adipogenic differentiation ability of the P8 generation iMSC cells prepared by Example 1 was further detected, as follows:

[0093] Osteogenic differentiation identification: P8 generation iMSC cells were inoculated into a cell culture container according to the appropriate inoculation density, and an appropriate amount of preheated fresh iMSC expansion culture medium was added, and shaken horizontally and crosswise three times, and placed in a 37°C, 5% CO2 concentration, and saturated humidity incubator, and shaken horizontally and crosswise three times again, and cultured; iMSCs were evenly spread and grew, and when the culture confluence reached 80%-90%, the culture medium in the container was aspirated, and osteogenic differentiation induction medium was started to be replaced, recorded as day 0, and the medium was replaced every three days, and the culture was continued to day 21; wherein the osteogenic differentiation induction medium used was Alpha-MEM medium containing 10% FBS, 1% Penicillin-Streptomycin, 10mM β-glycerophosphate (sodium β-glycerophosphate), 10nM Dexamethasone (dexamethasone), and 50μg / ml Ascorbic Acid (L-ascorbic acid). After day 21, the hard bone differentiated iMSCs were washed with pure water, and an appropriate volume of alizarin red working solution was added, and incubated at room temperature for 20-30min in the dark, then the excess dye was aspirated, and an appropriate volume of physiological saline or DPBS was added to each well, and observed under a microscope, and photographed, and the results are shown in Figure 4. According to the results shown in Figure 4, calcium nodules were obviously generated. Figure 4 Figure 4

[0094] ​​Adipogenic differentiation identification: P8 generation iMSC cells are inoculated into a cell culture container according to the appropriate inoculation density, and an appropriate amount of preheated fresh iMSC expansion culture medium is added; shake horizontally crosswise three times, place in a 37°C, 5% CO2 concentration, saturated humidity incubator, shake horizontally crosswise three times again, culture, iMSC evenly spread growth, when the culture confluence reaches 80%-90%, the culture medium in the container is aspirated and discarded, and the replacement of adipogenic differentiation induction medium is started, recorded as day 0, the medium is replaced every three days, and the culture is continued to day 21; wherein the adipogenic differentiation induction medium used is: Alpha-MEM medium containing 10% FBS, 1% Penicillin-Streptomycin, 1 μM Dexamethasone (dexamethasone), 0.5 mM IBMX (3-isobutyl-1-methyl xanthine), 0.2 mM Indomethacin (indomethacin), 10 ug / ml insulin; after day 21, the adipogenic differentiated iMSC is washed with normal saline or DPBS, and then the cells are washed with 60% isopropyl alcohol solution to prevent the residual normal saline or DPBS from causing the staining solution to precipitate; add an appropriate volume of oil red O working solution to the differentiation group and the control group, incubate at room temperature for 20-60 min in the dark, then aspirate and discard the excess staining solution, wash with normal saline or DPBS until no background color is observed, add an appropriate volume of normal saline or DPBS to each well for infiltration, observe under a microscope, and take a photo; the results are shown in Figure 4 According to the results shown in Figure 4 , lipid droplets are obviously formed.

[0095] Example 2

[0096] Based on Example 1, this embodiment further provides a method for obtaining an extracellular matrix of cell engineered human collagen by 3D culture of induced mesenchymal stem cells, and the specific steps are as follows:

[0097] I. The polyether sulfone 3D porous scaffold carrier is soaked in PBS, then sterilized using 120 kPa high-pressure steam at 121°C for 30 min to obtain a sterile polyether sulfone 3D porous scaffold carrier after sterilization; wherein the specific preparation method of the polyether sulfone 3D porous scaffold carrier can be referred to the patent document CN117551301A.

[0098] II. Using sterile tweezers, clamp the sterilized polyether sulfone 3D porous sponge-like scaffold carrier into the spinner flask, 70 carriers are placed in one spinner flask (30 million cells are plated on each carrier, so one spinner flask can plate 2100 million cells), after placing the carriers in the spinner flask, slightly shake the spinner flask to reduce the gap between the carriers; use a pipette to take 350 ml of diluted iMSC cell suspension (where the iMSC cells used here are P6 cells prepared in Example 2, the density of the cell suspension is 2x10 5

[0099] The 3D culture of the carrier is continued to the 15th day, and the mesenchymal stem cell expansion medium is replaced with an expression medium that can promote the generation of extracellular matrix. After replacing the expression medium, the medium is changed every 3 days, and the culture is continued for 15 days. A light yellow jelly-like extracellular matrix is generated on the carrier, and the culture is complete. The expression medium used in this step is: DMEM / F12 basic medium, 1% GlutaMax additive, 1% NEAA non-essential amino acid, 1% ITS-X supplement, 1% human blood albumin, 5 ng / ml EGF, 5 ng / ml bFGF, 5 ng / ml TGF-β, and 8 ng / ml PDGF-BB. The mesenchymal stem cell expansion medium used is: mesenchymal stem cell serum-free basic medium (the basic medium is selected from DMEM / F12 medium), 5% human platelet lysate, 50 mM ascorbic acid, 1% GlutaMax additive, 1% NEAA non-essential amino acid, 1% ITS-X supplement, 5 ng / ml IGF, 5 ng / ml bFGF, and 5 ng / ml PDGF-BB.

[0100] Example 2

[0101] (1) Extracellular matrix morphology:

[0102] In Example 2, the 3D carrier rich in collagen extracellular matrix is obtained by replacing the expression medium and continuing to culture for 15 days. A light yellow jelly-like extracellular matrix is generated on the carrier.

[0103] (2) Cell proliferation: ​

[0104] In the 3D culture performed in Example 2, one piece of the carrier at Day 5, Day 12, Day 20 and Day 25, respectively, was taken out and placed in a 6-well plate, and DAPI was used for staining to verify cell proliferation, with the specific steps as follows:

[0105] The carrier was washed once with PBS, and then the cells were fixed with 4% paraformaldehyde for about 10 minutes, and the fixing solution was removed and washed with washing solution for about 5 minutes each time; a small amount of nuclear staining solution DAPI was added, and staining was performed at room temperature for about 5 minutes; the DAPI staining solution was removed, and the washing solution was washed for about 3-5 minutes each time; and after washing, direct observation was performed under a fluorescence microscope. The observation results are shown in Figure 5 The DAPI staining of the cell nucleus is blue fluorescence, and cell proliferation is obvious.

[0106] Example 3

[0107] Based on Example 2, this example further provides a method for extracting the extracellular matrix of the cell-engineered collagen.

[0108] I. Decellularization of the sponge-shaped carrier

[0109] After the carrier obtained after the culture in Example 2 was taken, the expression culture medium in the roller bottle was discarded, 6 ml of water for injection was added along the wall of the roller bottle, the bottle cap was covered, the roller bottle was placed horizontally, and the roller bottle was slightly rotated to wash the residual culture medium on the carrier. Then, the water in the roller bottle was discarded, and a 400-mesh screen was used for filtration during discarding. The washing was repeated three times.

[0110] The decellularization solution was prepared, the pH of the decellularization solution was adjusted to 8-9, the prepared decellularization solution was added into the roller bottle along the wall (6 ml of decellularization solution was used for each carrier), and the decellularization was performed by vertical placement for 20 minutes. After the time ended, the decellularization solution in the roller bottle was discarded, a 400-mesh screen was used for filtration during discarding, and 6 ml of water for injection was added for repeated washing three times. A 400-mesh screen was used for filtration during washing. The decellularization operation of the carrier was completed. The formula of the decellularization solution used is as follows: the concentration of SDS is 0.1%, and the concentration of Triton X-100 is 1%. The preparation method of 100 mL of the decellularization solution containing 0.1% SDS and 1% Triton X-100 is as follows: 0.1 g of SDS was accurately weighed by an electronic balance and added into an appropriate amount of PBS, and a magnetic stirrer was used for stirring until complete dissolution. 1 mL of Triton X-100 was sucked by a pipette and added into the above solution, and stirring was continued until uniform. Finally, PBS was used for constant volume to 100 mL.

[0111] Using tweezers, the carrier in the flask was clamped out and placed in a 150 mm culture dish. The culture dish containing the carrier was first placed in a low-temperature freezer at -20°C, and after the carrier was completely frozen, the culture dish was placed in a freeze dryer for freeze drying. The freeze-dried carrier was obtained, and the completely freeze-dried carrier was fluffy, with filamentous protein attached to the carrier.

[0112] II. Extraction of the extracellular matrix of cell-engineered human collagen

[0113] The freeze-dried carrier obtained in step one was placed in a dry and anhydrous container, and the prepared DMSO-acetic acid solution (the DMSO-acetic acid solution was prepared in a volume ratio of 98:2, and 5 ml of the prepared DMSO-acetic acid solution was added to each carrier) was added. The culture dish was placed in a constant-temperature water bath shaker and shaken for 1 hour to allow the carrier to fully dissolve in the solution.

[0114] The dissolved solution was transferred to a centrifuge tube, and a high-speed centrifuge was used to centrifuge at 8000 rpm for 10 min. After centrifugation, a 400-mesh sieve was used to sieve to prevent protein from falling off due to pouring of the supernatant.

[0115] After pouring off the supernatant, the DMSO-acetic acid solution was continuously added to the precipitate for centrifugation until the supernatant was added to the injection water without white flocculent precipitate. If white flocculent precipitate was produced when the supernatant was added to the injection water, the DMSO-acetic acid solution was continuously added for centrifugation.

[0116] After confirming that there was no residual polyether sulfone in the solution, the precipitate was desalted using injection water at 8000 rpm until the conductivity of the supernatant was reduced to less than 15 us / cm. The carrier with qualified conductivity was used to extract the protein, and a high-pressure homogenizer was used to homogenize the protein solution at 1000 pa for 3 cycles. The homogenized protein solution was placed in a freeze-drying container, frozen at -20°C, and then transferred to a freeze dryer for freeze drying. Thus, the extracellular matrix of cell-engineered human collagen was obtained.

[0117] Effect Example 3

[0118] This effect example further detects the carrier protein extracted by the method in Example 3, and the specific steps are as follows:

[0119] An SDS-PAGE electrophoresis gel was configured. The freeze-dried extracellular matrix of cell-engineered human collagen was dissolved with an appropriate amount of sterile water to obtain a 3D carrier-cultured protein stock solution. The protein stock solution was concentrated using a 50-100 kDa ultrafiltration membrane to obtain a 3D carrier-cultured protein concentrate. After spotting, SDS-PAGE electrophoresis was performed. After electrophoresis, the gel was dyed and decolorized until the protein band was clear, and the dyed gel band was photographed.

[0120] The results are shown in Table 1. Figure 6 As shown in Table 1, according to the results shown in Table 1, it can be seen that the collagen I band with a size of 100-140 KDa is expressed. Figure 6

[0121] Comparative Example 1

[0122] Based on Example 2, this comparative example further investigates the influence of 3D culture of mesenchymal stem cells using different expression medium formulations on the expression level of collagen I in the final obtained extracellular matrix, as follows:

[0123] The following groups were set up, respectively:

[0124] Group 1: mesenchymal stem cell expansion medium → production medium No. 1 (add CHIR99021 + A8301) → expression medium; that is, mesenchymal stem cell expansion medium culture for 8 days, production medium No. 1 (add CHIR99021 + A8301) culture for 7 days, expression medium culture for 15 days; the difference from Example 2 is that production medium No. 1 (add CHIR99021 + A8301) is added for culture, and the mesenchymal stem cell expansion medium culture for 15 days is replaced by mesenchymal stem cell expansion medium culture for 8 days, production medium No. 1 (add CHIR99021 + A8301) culture for 7 days, and the rest is the same as Example 2;

[0125] Group 2: mesenchymal stem cell expansion medium → production medium No. 1 (add A8301 + Y-27632) → expression medium; that is, mesenchymal stem cell expansion medium culture for 8 days, production medium No. 1 (add A8301 + Y-27632) culture for 7 days, expression medium culture for 15 days; the difference from Example 2 is that production medium No. 1 (add A8301 + Y-27632) is added for culture, and the mesenchymal stem cell expansion medium culture for 15 days is replaced by mesenchymal stem cell expansion medium culture for 8 days, production medium No. 1 (add A8301 + Y-27632) culture for 7 days, and the rest is the same as Example 2;

[0126] Group 3: mesenchymal stem cell expansion medium → expression medium; that is, mesenchymal stem cell expansion medium culture for 15 days, expression medium culture for 15 days, the difference from Example 2 is that adipose mesenchymal stem cells are used instead of iMSC cells for culture, and the rest is the same as Example 2;

[0127] ​Group 4: Mesenchymal stem cell expansion medium → production No. 1 medium → expression medium; that is, mesenchymal stem cell expansion medium culture for 8 days, production No. 1 medium culture for 7 days, expression medium culture for 15 days, the difference from Example 2 is that the production No. 1 medium is added for culture, and the mesenchymal stem cell expansion medium culture for 15 days is replaced by mesenchymal stem cell expansion medium culture for 8 days, production No. 1 medium culture for 7 days, and the rest are the same as Example 2;

[0128] Group 5: Mesenchymal stem cell expansion medium → expression medium; that is, mesenchymal stem cell expansion medium culture for 15 days, expression medium culture for 15 days, same as Example 2;

[0129] Group 6: Mesenchymal stem cell expansion medium → expression medium (add CHIR99021 + A8301 + Y-27632); that is, mesenchymal stem cell expansion medium culture for 15 days, expression medium culture for 15 days, the difference from Example 2 is that the expression medium culture for 15 days is replaced by expression medium (add CHIR99021 + A8301 + Y-27632) culture for 15 days, and the rest are the same as Example 2;

[0130] Group 7: Mesenchymal stem cell expansion medium → expression medium (add A8301 + Y-27632); that is, mesenchymal stem cell expansion medium culture for 15 days, expression medium (add A8301 + Y-27632) culture for 15 days, the difference from Example 2 is that the expression medium culture for 15 days is replaced by expression medium (add A8301 + Y-27632) culture for 15 days, and the rest are the same as Example 2.

[0131] The medium formula used in the above experimental groups is as follows:

[0132] The production No. 1 medium formula is: Alpha-MEM basic medium, 3% human platelet lysate by volume concentration, 1% GlutaMax additive by volume concentration, 1% NEAA non-essential amino acids by volume concentration, 1% ITS-X supplement by volume concentration, 5 ng / ml IGF, 5 ng / ml EGF, 5 ng / ml bFGF, 5 ng / ml TGF-β;

[0133] Production Medium 1 (CHIR99021 + A8301) formulation: basal medium Alpha-MEM, human platelet lysate at 3% vol / vol, GlutaMax supplement at 1% vol / vol, NEAA non-essential amino acids at 1% vol / vol, ITS-X supplement at 1% vol / vol, IGF at 5 ng / ml, EGF at 5 ng / ml, bFGF at 5 ng / ml, TGF-beta at 5 ng / ml, CHIR99021 at 5 mM, A8301 at 5 mM;

[0134] Production Medium 1 (A8301 + Y-27632) formulation: basal medium Alpha-MEM, human platelet lysate at 3% vol / vol, GlutaMax supplement at 1% vol / vol, NEAA non-essential amino acids at 1% vol / vol, ITS-X supplement at 1% vol / vol, IGF at 5 ng / ml, EGF at 5 ng / ml, bFGF at 5 ng / ml, TGF-beta at 5 ng / ml, A8301 at 5 mM, Y-27632 at 8 mM;

[0135] Expression Medium formulation: basal medium DMEM / F12, GlutaMax supplement at 1% vol / vol, NEAA non-essential amino acids at 1% vol / vol, ITS-X supplement at 1% vol / vol, human albumin at 1% vol / vol, EGF at 5 ng / ml, bFGF at 5 ng / ml, TGF-beta at 5 ng / ml, PDGF-BB at 8 ng / ml;

[0136] Expression Medium (CHIR99021 + A8301 + Y-27632) formulation: basal medium DMEM / F12, GlutaMax supplement at 1% vol / vol, NEAA non-essential amino acids at 1% vol / vol, ITS-X supplement at 1% vol / vol, human albumin at 1% vol / vol, EGF at 5 ng / ml, bFGF at 5 ng / ml, TGF-beta at 5 ng / ml, PDGF-BB at 8 ng / ml, CHIR99021 at 5 mM, A8301 at 5 mM, Y-27632 at 8 mM;

[0137] 3D Medium (Expression Medium) (A8301 + Y-27632) formulation: basal medium DMEM / F12, GlutaMax supplement at 1% vol / vol, NEAA non-essential amino acids at 1% vol / vol, ITS-X supplement at 1% vol / vol, human albumin at 1% vol / vol, EGF at 5 ng / ml, bFGF at 5 ng / ml, TGF-beta at 5 ng / ml, PDGF-BB at 8 ng / ml, A8301 at 5 mM, Y-27632 at 8 mM.

[0138] Based on the protein extraction method in Example 3, the extracellular matrix extracted from the 3D culture obtained by culturing mesenchymal stem cells in the above group manner was subjected to cell extracellular matrix extraction, and the corresponding carrier protein solution was obtained; after the protein solution obtained in each group was subjected to flow-through and rinsing, the protein (Collagen I) concentration (mg / mL) and the total amount of protein (Collagen I) obtained in each group were detected, and the specific detection method was as follows: the protein concentration was detected using a BCA detection kit, and the total amount of protein was weighed after freeze-drying, and the results are shown in Table 2; at the same time, based on the same method as in Effect Example 3, the obtained carrier protein was subjected to SDS-PAGE electrophoresis, and the results are shown in Table 2 and Figure 7 、 Figure 8

[0139] Table 2 Protein concentration (mg / mL) and total protein amount (mg) results

[0140]

[0141] Based on the results shown in Table 2 and Figure 7 、 Figure 8 , the protein content in the extracellular matrix obtained by using the specific preparation method of the fifth group: mesenchymal stem cell expansion medium→3D culture medium (i.e., Example 2) is the highest among all groups. Compared with the fifth group (Example 2), the first group, the second group, and the fourth group, the iMSC cells were cultured by increasing the production of culture medium No. 1 and changing the medium replacement time node during the culture process, but the protein expression in the obtained extracellular matrix was significantly lower. At the same time, compared with the fifth group (Example 2), the sixth group and the seventh group, the iMSC cells were cultured by changing the specific formula of the expression medium, and the protein expression in the obtained extracellular matrix was also significantly lower. At the same time, compared with the fifth group (Example 2), the third group replaced the iMSC cells with adipose mesenchymal stem cells for the culture and preparation of extracellular matrix, and it was found that the iMSC differentiated from iPSC was superior to adipose mesenchymal stem cells in producing extracellular matrix rich in type I collagen. Therefore, in the scheme of preparing extracellular matrix by 3D culture, the specific culture medium used, the culture method used, and the source of mesenchymal stem cells all have a significant impact on the protein expression in the finally prepared extracellular matrix. According to the above results, it is further shown that the preparation of extracellular matrix by the 3D culture method provided in the present application can make the prepared extracellular matrix rich in collagen, and the type I collagen in the extracellular matrix has a high expression amount. This further shows that the preparation method provided in the present application has a significant advantage in promoting the generation of type I collagen in the extracellular matrix.​

[0142] It is to be understood that the present application is described by way of example only, and that modifications or alterations can be made to the features and embodiments described without departing from the spirit and scope of the application. In addition, modifications can be made to the features and embodiments described to accommodate specific situations and materials without departing from the spirit and scope of the application. Accordingly, the application is not limited to the specific embodiments disclosed herein, but rather, the scope of the application includes all embodiments falling within the scope of the claims.

Claims

1. A method for preparing an extracellular matrix of cell-engineered human collagen, characterized in that, Includes the following steps: S1: Culture and directly induce differentiation of human induced pluripotent stem cells or cell cultures including human induced pluripotent stem cells to obtain induced mesenchymal stem cells or cell cultures including induced mesenchymal stem cells. S2: The induced mesenchymal stem cells are cultured in 3D using a 3D porous scaffold carrier; The 3D culture includes: dropping the induced mesenchymal stem cell suspension onto a 3D porous scaffold carrier, immersing the 3D porous scaffold carrier in the induced mesenchymal stem cell suspension; sequentially culturing the cells in a mesenchymal stem cell expansion medium and an expression medium; the expression medium contains the following components: basal medium and additives; the additives contain the following components: GlutaMax additive, NEAA non-essential amino acids, ITS-X supplement, human serum albumin, EGF, bFGF, TGF-β, and PDGF-BB.

2. The preparation method according to claim 1, characterized in that, The volume concentration of the GlutaMax additive is 0.5–2%; the volume concentration of the NEAA non-essential amino acids is 0.5–2%; the volume concentration of the ITS-X supplement is 0.5–2%; the volume concentration of the human serum albumin is 0.5–2%; the concentration of EGF is 1–10 ng / ml; the concentration of bFGF is 1–10 ng / ml; the concentration of TGF-β is 1–10 ng / ml; and the concentration of PDGF-BB is 1–10 ng / ml. Preferably, the basal culture medium is selected from any one or more of high-glucose DMEM, Alpha-MEM, and DMEM / F12 culture medium.

3. The preparation method according to claim 1, characterized in that, In step S2, the 3D culture includes: The induced mesenchymal stem cell suspension was dropwise onto a sterilized 3D porous scaffold carrier, immersing the 3D porous scaffold carrier in the induced mesenchymal stem cell suspension to obtain a mixed system of scaffold and cell suspension. Mesenchymal stem cell expansion medium was added to the mixed system for a second 3D culture. After the second 3D culture, the mesenchymal stem cell expansion medium was replaced with expression medium for a third 3D culture. Preferably, the time for the second 3D culture is 10-20 days; the time for the third 3D culture is 10-20 days. Preferably, the culture medium is changed every 3 to 5 days during the secondary and tertiary 3D culture processes; Preferably, the 3D culture conditions are a temperature of 35-40℃ and a CO2 concentration of 2-6%.

4. The preparation method according to claim 1, characterized in that, In step S2, the 3D culture includes: The sterilized 3D porous scaffold carrier is clamped into a roller bottle, and the induced mesenchymal stem cell suspension is dropped onto the sterilized 3D porous scaffold carrier, so that the 3D porous scaffold carrier is immersed in the induced mesenchymal stem cell suspension. Place the roller bottle in an incubator for a single 3D culture; Add mesenchymal stem cell expansion medium to the roller bottle after one 3D culture, and place the roller bottle in a roller bottle culture machine for a second 3D culture. After a second 3D culture, the mesenchymal stem cell expansion medium was replaced with expression medium, and the roller bottle was placed in a roller bottle culture machine for a third 3D culture. Preferably, the duration of the first 3D culture is 1-5 hours; the duration of the second 3D culture is 10-20 days; and the duration of the third 3D culture is 10-20 days. Preferably, the 3D culture conditions are a temperature of 35-40℃ and a CO2 concentration of 2-6%. Preferably, the culture medium is changed every 3 to 5 days during the secondary and tertiary 3D culture processes; Preferably, 30 to 70 of the 3D porous support carriers are placed in a single rotating bottle.

5. The preparation method according to any one of claims 1, 3-4, characterized in that, The density of the induced mesenchymal stem cell suspension is (2.0–3.0) x 10⁻¹⁰. 5 cells / ml; preferably, the induced mesenchymal stem cells used are P4 to P12 generation induced mesenchymal stem cells.

6. The preparation method according to any one of claims 1, 3-4, characterized in that, The mesenchymal stem cell expansion culture medium contains the following components: mesenchymal stem cell basal culture medium and additives, wherein the additives include: human platelet lysate, ascorbic acid, GlutaMax additive, NEAA non-essential amino acids, ITS-X supplement, IGF, bFGF, and PDGF-BB. Preferably, the additive comprises the following components: human platelet lysate at a volume concentration of 1-10%, 30-70 mM ascorbic acid, GlutaMax additive at a volume concentration of 0.5%-2%, NEAA non-essential amino acids at a volume concentration of 0.5%-2%, ITS-X supplement at a volume concentration of 0.5%-2%, 1-10 ng / ml IGF, 1-10 ng / ml bFGF, and 1-10 ng / ml PDGF-BB; Preferably, the mesenchymal stem cell basal culture medium is selected from serum-free mesenchymal stem cell basal culture medium; the serum-free mesenchymal stem cell basal culture medium is selected from at least one of high glucose DMEM, Alpha-MEM, and DMEM / F12 culture medium.

7. The preparation method according to claim 1, characterized in that, The preparation method further includes: S3: Decellularize the vector obtained after 3D culture in step S2; S4: Extraction of the extracellular matrix of cell-engineered human collagen; Preferably, the decellularization process includes: Take the vector obtained after 3D culture, remove the culture medium, and wash with water for injection; Add decellularization solution to the cleaned carrier for decellularization treatment, wash after decellularization treatment, and then freeze-dry. Preferably, the decellularization solution comprises the following components: SDS with a mass concentration of 0.1%-1% and Triton X-100 with a mass concentration of 0.5%-2%.

8. The preparation method according to claim 7, characterized in that, The extraction includes: Add DMSO-acetic acid solution to the freeze-dried carrier to dissolve the carrier in the DMSO-acetic acid solution, and then centrifuge. Add DMSO-acetic acid solution to the precipitate obtained after centrifugation, and continue centrifuging. Repeat this process until no white flocculent precipitate is produced when the supernatant is added to water for injection. The final precipitate was desalted, homogenized, and freeze-dried to obtain an extracellular matrix rich in cell-engineered human collagen; Preferably, the DMSO-acetic acid solution is prepared by mixing DMSO and acetic acid in a ratio of 95-99:0.5-5; preferably, the centrifugation conditions are centrifugation at 6000-10000 rpm for 5-15 min; the desalting conditions are desalting until the conductivity of the supernatant drops below 15 μS / cm; preferably, the homogenization conditions are 800-1500 Pa, and the number of homogenizations is 2-4.

9. An extracellular matrix of cell-engineered human collagen prepared by the preparation method according to any one of claims 1-8; preferably, the cell-engineered human collagen is type I collagen.

10. An application of the extracellular matrix of cell-engineered human collagen as described in claim 9 in the field of biomaterial preparation; preferably, the application includes applications in the fields of biomedical materials preparation and cosmetic skincare products; preferably, the biomedical materials include at least one of tissue engineering scaffolds, wound dressings, and hemostatic materials; preferably, the cosmetic skincare products include at least one of skincare products and cosmetic injections.

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