Decellularized scaffold as well as preparation method and application thereof

Abstract

The invention discloses a decellularized scaffold as well as a preparation method and application thereof. The decellularized scaffold provided by the invention can effectively support in-vitro growth and development of follicles. The material of the decellularized scaffold is completely derived from itself, and subsequent immune transplantation treatment is completely not needed. After in-vitro follicle planting and induced culture are completed, the decellularized scaffold can be planted back into a body again, and the reproductive endocrine function and the fertility are recovered. According to the invention, powerful material support is provided for large-scale safe application of artificial ovaries, and a new choice is provided for retention of fertility functions.

Description

Technical Field

[0001] The invention relates to a cell scaffold in the fields of biomedical engineering, follicle culture technology and clinical reproductive medicine, and specifically relates to a decellularized scaffold and a preparation method and application thereof. Background Art

[0002] Cryopreserved ovarian tissue transplantation has become a key technology in the field of reproductive medicine. Although there have been reports of live births from ovarian tissue transplantation and the technology is becoming increasingly mature, some defects have hindered its wider application. Key issues include the risk of malignant tumor recurrence and the lack of natural, non-immune scaffold materials for follicle reimplantation. The development of tissue engineering technology has made it possible to invent new scaffolds for this type of ovarian transplantation. Good sources of scaffold materials must tend to be selected from natural autologous extracellular matrix materials, because such materials are similar in composition and structure to the original ovarian tissue, and can simultaneously solve the problems of donor shortage and immune rejection. Extracellular matrix materials contain a variety of growth factors and active factors that support cell growth and differentiation, can induce the growth, migration and differentiation of implanted cells, and can also provide a suitable growth environment for new blood vessels.

[0003] Decellularized scaffolds are a technology that removes cellular components from target tissues, leaving behind the ECM. This technology can maximize the retention of the natural biochemical properties and structural integrity of the extracellular part of the tissue. Decellularized biological scaffolds have multiple advantages, including the retention of adhesion molecules and extracellular factors necessary for cell adhesion, proliferation, differentiation, and angiogenesis. The decellularized ECM maintains the complex spatial and mechanical properties of the native tissue and can fully adapt to the highly dynamic and specific process of follicle development. Due to the limited source of decellularized scaffolds in the ovary itself, allogeneic donated ovarian tissues pose risks such as potential pathogen transmission and immune rejection. There is currently no precedent for reliable autologous non-ovarian decellularized scaffolds to support follicle development. Therefore, the use of autologous non-ovarian tissues to provide sufficient and suitable scaffolds to support follicle growth remains an important research area.

[0004] Patent CN112717201A discloses an ovarian extracellular matrix scaffold and hydrogel and its preparation method and application. However, the scaffold has certain defects in actual implementation and will also face certain limitations in large-scale application. First, the source of ovarian extracellular matrix scaffold is relatively limited. If xenogeneic or allogeneic ovarian tissue is used, it may cause a series of problems such as ethical issues, potential pathogen transmission risks and immune rejection. Secondly, the natural extracellular matrix itself has a complex protein structure, and in the process of freeze-drying, grinding into particles with a particle size of less than 1mm and mixing with acid and pepsin, the arrangement structure and biological activity of the extracellular matrix will be destroyed, thereby affecting the signal transduction between the follicle and its microenvironment. Finally, the extracellular matrix has unique mechanical properties. The follicle can sense and regulate the mechanical changes of the surrounding matrix through growth and volume changes. However, it is difficult for hydrogels to completely simulate and replace this dynamic mechanical adaptability.

[0005] Therefore, the existing follicle culture scaffold technology lacks efficient scaffold materials that do not require immunosuppressive treatment, resulting in low efficiency of follicle growth and maturation in vitro. Summary of the invention

[0006] In order to overcome the problems and needs existing in the background technology, the present invention provides a decellularized scaffold and its preparation method and application. The decellularized scaffold proposed in the present invention can effectively support the growth and development of follicles in vitro. The decellularized scaffold material is completely derived from the body itself and does not require subsequent immune transplantation treatment. After completing the in vitro follicle planting and induced culture, the above-mentioned scaffold material can be re-implanted into the body to restore reproductive endocrine function and fertility. The present invention provides strong material support for the large-scale and safe application of artificial ovaries and provides a new option for retaining fertility function.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] 1. A decellularized scaffold for follicle development

[0009] The decellularized scaffold is prepared by the following preparation method:

[0010] 1) Take the ovarian surrounding tissue of a mammal on a clean bench and record it as the tissue to be processed, which is inactive. After removing impurities and slicing the tissue to be processed, obtain tissue slices;

[0011] 2) Multiple decellularization treatments, including:

[0012] 2.1) Wash tissue sections with double distilled water and NaCl solution in sequence to separate cells from the surrounding extracellular matrix;

[0013] 2.2) using an ultra-low temperature refrigerator and an incubator to perform multiple freezing and thawing processes on the tissue sections processed in 2.1);

[0014] 2.3) washing the tissue sections treated in 2.2) with sodium dodecyl sulfate solution and PBS solution in sequence, so that the nuclei and cytoplasmic components inside the cells are completely released;

[0015] 2.4) The tissue sections treated in 2.3) are incubated in DNA enzyme and lipase for 24 hours, so that the nucleic acid and fat components in the tissue sections are removed, thereby preparing a decellularized scaffold, and then washed with PBS solution for multiple times (such as 3 times, 30 minutes each time), and finally stored at -80°C for future use. The decellularized scaffold is in the form of a thin sheet, and is formed by spreading it on the bottom of a sterile culture dish.

[0016] In the above 1), the size of the tissue slice is 3-5 mm×3-5 mm×1-2 mm.

[0017] The 2.1) is specifically:

[0018] First, wash the tissue sections with double distilled water (ddH2O) on a shaker for several times (e.g., 3 times, 30 minutes each time); then, wash the tissue with 10% NaCl solution for several hours (e.g., 2 hours); then, wash the tissue sections with double distilled water (ddH2O) on a shaker for several times (e.g., 3 times, 30 minutes each time); finally, wash the tissue sections with PBS solution on a shaker for several times (e.g., 3 times, 30 minutes each time). This process mainly utilizes the change in osmotic pressure around cells to make fat and peritoneal tissue cells experience the process of absorbing water, increasing volume, dehydrating, and reducing volume, thereby achieving the purpose of separating cells from the surrounding extracellular matrix.

[0019] The 2.2) is specifically:

[0020] 2.1) The treated tissue sections are sequentially frozen in an ultra-low temperature freezer at -80°C and thawed in an incubator at 37°C, and repeated (e.g., three times). At the rapid freezing rate, ice crystals form in the cells in the tissue and destroy the cell structure.

[0021] The ovarian surrounding tissue is one or more of adipose tissue and abdominal wall peritoneal tissue.

[0022] The 2.3) is specifically:

[0023] The tissue slices treated in 2.2) are immersed in 4 times the volume of 1% SDS and washed for several hours (e.g., 4 hours) at room temperature with sufficient shaking, and the SDS is replaced every hour; then washed with PBS solution at room temperature for 3 times, 30 minutes each time, so that the remaining SDS is removed. The principle is to use the affinity of SDS molecules for hydrophobicity to dissolve the cell membrane and release the nucleus and cytoplasm components inside the cell.

[0024] The 1% SDS is a double distilled aqueous solution of sodium dodecyl sulfate.

[0025] The shaker was a sterile shaker with a temperature of 37° C. and a stirring speed of 100 rpm.

[0026] The DNA enzyme is DNase, and the concentration is 100-200 U / mL.

[0027] The concentration of the lipase is 5 U / mL.

[0028] 2. An artificial ovary complex

[0029] The artificial ovary complex is obtained by implanting follicles and ovarian stromal cells into the decellularized scaffold, and the artificial ovary complex has the function of repairing ovarian damage and reconstructing ovarian function in vitro. The preparation process of the artificial ovary complex specifically includes:

[0030] The decellularized scaffold is pre-soaked in αMEM culture medium for several minutes (e.g., 30 min), and follicles with a diameter of 100-150 μm, intact basement membrane, central spherical oocytes, and surrounding granulosa cells (GCs) are selected for scaffold implantation experiments in the decellularized scaffold to construct an artificial ovarian complex.

[0031] 3. A method for preparing a decellularized scaffold for follicle development

[0032] 1) Take the ovarian surrounding tissue of a mammal on a clean bench and record it as the tissue to be processed, which is inactive. After removing impurities and slicing the tissue to be processed, obtain tissue slices;

[0033] 2) Multiple decellularization treatments, including:

[0034] 2.1) Wash tissue sections with double distilled water and NaCl solution in sequence to separate cells from the surrounding extracellular matrix;

[0035] 2.2) using an ultra-low temperature refrigerator and an incubator to perform multiple freezing and thawing processes on the tissue sections processed in 2.1);

[0036] 2.3) washing the tissue sections treated in 2.2) with sodium dodecyl sulfate solution and PBS solution in sequence, so that the nuclei and cytoplasmic components inside the cells are completely released;

[0037] 2.4) The tissue sections treated in 2.3) are incubated in DNA enzyme and lipase for 24 hours, so that the nucleic acid and fat components in the tissue sections are removed, thereby preparing a decellularized scaffold, and then washed with PBS solution for multiple times (such as 3 times, 30 minutes each time), and finally stored at -80°C for future use. The decellularized scaffold is in the form of a thin sheet, and is formed by spreading it on the bottom of a sterile culture dish.

[0038] The 2.1) is specifically:

[0039] First, wash the tissue sections with double distilled water (ddH2O) on a shaker for several times (e.g., 3 times, 30 minutes each time); then, wash the tissue with 10% NaCl solution for several hours (e.g., 2 hours); then, wash the tissue sections with double distilled water (ddH2O) on a shaker for several times (e.g., 3 times, 30 minutes each time); finally, wash the tissue sections with PBS solution on a shaker for several times (e.g., 3 times, 30 minutes each time). This process mainly utilizes the change in osmotic pressure around cells to make fat and peritoneal tissue cells experience the process of absorbing water, increasing volume, dehydrating, and reducing volume, thereby achieving the purpose of separating cells from the surrounding extracellular matrix.

[0040] IV. Application of a decellularized scaffold in the preparation of products for ovarian damage and reconstruction of ovarian function

[0041] The ovarian damage includes any one of ovarian damage caused by aging, ovarian damage caused by genetic or immune function defects or environment, iatrogenic ovarian damage or pathological ovarian damage.

[0042] Among them, patients undergoing tumor treatment benefit the most, because this technology can minimize the risk of recurrence caused by residual tumor cells, and subsequent implantation can restore ovarian function without the need for further immune transplantation treatment.

[0043] The beneficial effects of the present invention are mainly reflected in the following aspects:

[0044] 1. The preparation method proposed in the present invention can retain the extracellular matrix components of fat and peritoneum to the greatest extent possible, and the active ingredients are close to the ovarian decellularized matrix components, which can provide a growth environment closer to the natural one for follicle development, that is, it can provide a better growth environment for follicles.

[0045] 2. The decellularized scaffold proposed in the present invention is derived from one's own fat and peritoneal tissue, and has no possibility of immune rejection. It is not limited by the shortage of allogeneic donor tissue and does not require subsequent immunosuppressive treatment.

[0046] 3. After the follicles are implanted into the decellularized scaffold of the present invention, the decellularized scaffold material implanted with the follicles can secrete estrogen, progesterone, and inhibin in the in vitro culture medium, and can be re-implanted into the body by minimally invasive laparoscopy or a small subcutaneous incision, providing a good growth environment for the growth of the follicles in vivo, and providing a new option for restoring reproductive endocrine and fertility functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a real picture of the fat and peritoneal cell decellularized scaffold prepared by the present invention.

[0048] Figure 2 Pathological staining images of rat fat and peritoneal tissue before and after decellularization in an embodiment of the present invention.

[0049] Figure 3 This is an immunohistochemical expression diagram of the adipose and peritoneal extracellular matrix in the examples of the present invention.

[0050] Figure 4 This is a view of adipose and peritoneal decellularized matrix under a scanning electron microscope according to an embodiment of the present invention.

[0051] Figure 5 The growth of the ovarian follicles implanted in the adipose and peritoneal decellularized matrix under an inverted microscope according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to better understand the present invention, and thus to more clearly define the scope of protection claimed in the present invention, the present invention is described in detail with respect to certain specific embodiments and drawings of the present invention. It should be noted that the following are only certain specific implementation methods of the present invention, which are only part of the embodiments of the present invention, wherein the specific and direct description of the relevant structures is only for the convenience of understanding the present invention, and each specific feature does not naturally and directly limit the scope of implementation of the present invention. The conventional selections and substitutions made by those skilled in the art under the guidance of the present invention, as well as the reasonable arrangement and combination of several technical features under the guidance of the present invention, should all be deemed to be within the scope of protection claimed in the present invention.

[0053] 1. Definition and Use of Terms

[0054] The above-mentioned "ovarian function damage" includes damage caused by any one or more of age, genetics, immunity, environmental factors, iatrogenic factors, and pathological factors. The specific results of the damage include irregular menstruation, infertility, amenorrhea, perimenopausal symptoms, etc.

[0055] The above-mentioned "reconstruction of ovarian function" includes the restoration of normal reproductive endocrine function and fertility, and the patient shows normal menstrual cycle or normal follicle development cycle.

[0056] The above-mentioned "follicle" refers to the basic functional unit of oocyte occurrence and development in the ovary of mammals, which is round and surrounded by granulosa cells and located in the ovarian cortex. It includes primordial follicles, primary follicles, and secondary follicles.

[0057] The above-mentioned "mammal" includes but is not limited to rats, mice, non-human primates, humans, dogs, cats, horses, cattle, sheep, pigs, goats, preferably humans, rats or pigs.

[0058] The above-mentioned "implantation" methods include but are not limited to minimally invasive laparoscopic or subcutaneous small incision transplantation.

[0059] 2. Experimental Materials

[0060] Double distilled water: prepared in the laboratory;

[0061] 10% NaCl solution: Hubei Tiansheng Pharmaceutical Co., Ltd., product number: National Medicine Standard H42021838;

[0062] PBS solution: Biosharp, catalog number: PBS BL302A;

[0063] Sodium dodecyl sulfate: Macklin, Product No.: S850167, C12H26O4S·Na

[0064] DNase: Macklin, catalog number: D875698;

[0065] Lipase: Macklin, product number: L812480-10g;

[0066] α-MEM medium: Macklin, catalog number: M917566;

[0067] CO2 incubator: New Brunswick, catalog number: Galaxy 170s;

[0068] Sterile shaker product number: Hangzhou Fisher, product number: NMYC-60C;

[0069] Collagenase type IV: Macklin, catalog number: C909791-100mg;

[0070] ITS additives: Sigma-Aldrich, catalog number: I3146-5ML;

[0071] Follicle stimulating hormone: Livzon Pharmaceutical Factory of Livzon Group, item number: National Medicine Standard H20052130

[0072] Fetal bovine serum: Macklin, catalog number: F917980;

[0073] Penicillin-Streptomycin: Thermo Fisher, catalog number: 15140-122.

[0074] Example 1

[0075] The present invention provides a method for preparing a decellularized scaffold, comprising the following steps:

[0076] 1) Pretreatment of fat and peritoneal tissue: Take the periovarian fat tissue and abdominal peritoneal tissue of mammals on a clean bench and record them as the tissue to be processed. Wash the tissue to be processed with PBS to remove impurities. Use a sterile blade to slice the tissue into thin slices of about 5mm×5mm×1-2mm in a low-temperature frozen state. After slicing, start the sterile decellularization process.

[0077] Among them, peritoneal tissue is peritoneal tissue of the inner wall of the abdominal cavity close to the level of the ovaries. The cryogenic freezing state is to maintain the fat and peritoneal tissue below 0°C in a solid state. The thickness of the fat and peritoneal tissue is controlled between 1-2mm. This thickness of tissue is most suitable for completing the decellularization process within the established time and procedure. At the same time, ophthalmic forceps and ophthalmic fine shears can be used to remove larger blood vessels and blood clots impurities from fat and peritoneal tissue during the pretreatment process to improve the decellularization effect of subsequent processes.

[0078] 2) Multiple decellularization: Take the fat and peritoneal tissues treated in step 1, first, wash the tissues with ddH2O on a shaker for 30 minutes, repeat 3 times. Each wash needs to be replaced with a new ddH2O washing solution, and the purpose of repeated washing is to remove residual blood, blood clots and lymph as much as possible. Then, wash the tissue with 10% NaCl solution for 2 hours, and then place it on a sterile shaker of ddH2O and PBS for 3 times, each time for 30 minutes. The purpose of sequential washing with 10% NaCl solution and ddH2O is to change the osmotic pressure around the cells, so that the cells are first dehydrated and then expanded and ruptured, which is more conducive to the subsequent washing process. The fat and peritoneal tissue slices are further frozen and thawed three times at -80°C and 37°C, respectively. Then immerse in 4 times the volume of 1% SDS and fully shake and wash at room temperature for 4 hours, and replace SDS every hour. The purpose of multiple washings with sodium dodecyl sulfate solution is to fully remove the cell structure in the tissue, such as nucleic acids, lipids and other components, and flush the fragmented cell structure out of the original tissue, and retain the composition and framework of the extracellular matrix as completely as possible. The tissue slices were washed again with PBS for 3 times at room temperature, each time for 30 minutes, to remove the remaining SDS. Finally, the tissue slices were incubated with DNase and lipase for 24 hours to obtain adipose and peritoneal decellularized scaffolds. Figure 1As shown. The purpose of using DNase and lipase to treat the decellularized scaffold is to further remove the DNA and fat components in the scaffold. Wash with PBS 3 times, 30 minutes each time. Store at -80℃ for later use.

[0079] Wherein, 1% SDS is sodium dodecyl sulfate solution, which is a double distilled water solution of sodium dodecyl sulfate, and the mass volume ratio between the sodium dodecyl sulfate and double distilled water is 1%. The environment is a sterile shaker, the temperature is 37°C, and the stirring speed is controlled to be 100rpm. The concentration of the DNase is 100U / mL. The concentration of the lipase is 5U / mL.

[0080] The present invention also proposes an artificial ovarian complex, which is obtained by implanting follicles and ovarian stromal cells into a decellularized scaffold. The artificial ovarian complex has the function of repairing ovarian damage and reconstructing ovarian function in vitro. Specifically, it includes:

[0081] The decellularized scaffold is pre-soaked in αMEM culture medium for several minutes (such as 30 minutes), wherein the α-MEM culture medium contains fetal bovine serum, ITS, follicle stimulating hormone and penicillin-streptomycin mixture. The volume percentage of fetal bovine serum is 10%, the volume percentage of the penicillin-streptomycin mixture is 1%, the working concentration of penicillin in the culture medium is 100U / ml, and the working concentration of streptomycin is 0.1mg / ml. The concentration of follicle stimulating hormone is 0.15mIU / mL. The ITS is composed of insulin, transferrin and sodium selenite, and the concentrations in the culture medium are 5μg / mL of insulin, 5μg / mL of transferrin and 5ng / mL of sodium selenite. Follicles with a diameter of 100-150μm, intact basement membrane, central spherical oocytes, and granulosa cells (GCs) around them are selected for scaffold implantation experiments in the decellularized scaffold, thereby constructing an artificial ovarian complex. The implantation density of the follicles is 30 follicles per 1cm 2 Adipose or peritoneal decellularized scaffolds.

[0082] After 72 hours of follicle implantation in adipose and peritoneal decellularized matrix, immunohistochemistry was performed to evaluate the growth of follicles in the scaffold. When the medium was changed every other day, the culture medium was taken to detect the secretion of estrogen, progesterone and inhibin.

[0083] Sodium dodecyl sulfate (SDS) is an anionic detergent that effectively dissolves cellular components while preserving the extracellular matrix (ECM) structure. After SDS-based decellularization, all three tissue scaffolds (ovary, adipose, and peritoneum) became translucent, indicating that the cellular components of the tissues were successfully removed while preserving the original ECM scaffold.

[0084] Combination Figure 2 , Figure 2A is the HE staining image of normal adipose tissue. Figure 2 B is the HE staining image of normal peritoneal tissue. Figure 2 C is the HE staining image of adipose tissue after decellularization. Figure 2 D is the HE staining image of peritoneal tissue after decellularization. Figure 2 In the HE staining results of C and D, it can be clearly seen that the cell structure of fat and peritoneal tissues has completely disappeared, and the extracellular matrix structure is intact. The extracellular matrix in fat and peritoneal decellularized tissues has a similar spatial structure as that in ovarian decellularized tissues.

[0085] Combination Figure 3 It can be proved that the decellularization method designed by the present invention has high efficiency in removing cells. Immunofluorescence examination of type I collagen, type IV collagen, fibronectin and laminin in the two decellularized scaffolds of fat and peritoneum are similar to the state before decellularization, indicating that this method has the greatest degree of retention of the extracellular matrix components in the original tissue. The major histocompatibility antigen type I and type II are not expressed in the two decellularized scaffolds, indicating that the elution process removes the immunogenicity of the tissue.

[0086] Combination Figure 4 Known fat ( Figure 4 B) and peritoneal decellularized scaffolds ( Figure 4 C) has the same characteristics as the ovarian decellularized scaffold under scanning electron microscopy ( Figure 4 A) have very similar ultrastructure.

[0087] Combination Figure 5 It can be seen that after the same number of follicles were implanted in three types of decellularized scaffolds, fat and peritoneum, the immunofluorescence expression of estrogen receptors and follicle-stimulating hormone receptors expressed by the follicles was similar ( Figure 5 A), progesterone secreted by the culture medium ( Figure 5 B) and inhibin ( Figure 5 The results show that the acellular scaffold prepared by the present invention can provide a good living environment for the growth of follicles, thereby achieving the purpose of repairing ovarian damage and reconstructing ovarian function.

[0088] Example 2

[0089] The present invention provides a method for preparing a SD rat decellularized scaffold, comprising the following steps:

[0090] 1) Pretreatment of fat and peritoneal tissue: Obtain the periovarian fat tissue of SD rats on a clean bench and record it as the tissue to be processed. Wash the tissue to be processed thoroughly with PBS to remove impurities. Use a sterile blade to slice the tissue into slices of about 5 mm × 5 mm × 1 mm in a low-temperature frozen state. After slicing, start the sterile decellularization process.

[0091] Among them, peritoneal tissue is peritoneal tissue of the inner wall of the abdominal cavity close to the level of the ovaries. The cryogenic freezing state is to maintain the fat and peritoneal tissue below 0°C in a solid state. The thickness of the fat and peritoneal tissue is controlled to be about 1 mm. This thickness of tissue is most suitable for completing the decellularization process within a given time and procedure. At the same time, ophthalmic forceps and ophthalmic fine shears can be used to remove larger blood vessels and blood clots impurities from fat and peritoneal tissue during the pretreatment process to improve the decellularization effect of subsequent processes.

[0092] 2) Multiple decellularization: Take the fat and peritoneal tissues treated in step 1), first, wash the tissues with ddH2O on a shaker for 30 minutes, repeat 3 times. Each wash needs to be replaced with a new ddH2O washing solution, and the purpose of repeated washing is to remove residual blood, blood clots and lymph as much as possible. Then, wash the tissue with 10% NaCl solution for 2 hours, and then place it on a sterile shaker of ddH2O and PBS for 3 times, each time for 30 minutes. The fat and peritoneal tissue slices are further frozen and thawed three times at -80°C and 37°C, respectively. Then immerse in 4 times the volume of 1% SDS and shake and wash at room temperature for 4 hours, and replace SDS every hour. Wash again with PBS at room temperature for 30 minutes each time to remove the remaining SDS. Finally, incubate the tissue slices with DNase and lipase for 24 hours to obtain a fat decellularized scaffold. Wash with PBS 3 times, each time for 30 minutes. Store at -80°C for later use.

[0093] Wherein, 1% SDS is sodium dodecyl sulfate solution, which is a double distilled water solution of sodium dodecyl sulfate, and the mass volume ratio between the sodium dodecyl sulfate and double distilled water is 1%. The environment is a sterile shaker, the temperature is 37°C, and the stirring speed is controlled to be 100rpm. The concentration of the DNase is 100U / mL. The concentration of the lipase is 5U / mL.

[0094] The present invention also proposes an artificial ovary complex of SD rats, which is obtained by implanting follicles and ovarian stromal cells into a decellularized scaffold. The artificial ovary complex has the function of repairing ovarian damage and reconstructing ovarian function in vitro. Specifically, it includes:

[0095] The decellularized scaffold was pre-soaked in αMEM culture medium for 30 minutes, wherein the α-MEM culture medium contained fetal bovine serum, ITS, follicle stimulating hormone and penicillin-streptomycin mixture. The volume percentage of fetal bovine serum was 10%, the volume percentage of the penicillin-streptomycin mixture was 1%, the working concentration of penicillin in the culture medium was 100U / ml, and the working concentration of streptomycin was 0.1mg / ml. The concentration of follicle stimulating hormone was 0.15mIU / mL. The ITS was composed of insulin, transferrin and sodium selenite, and the concentrations in the culture medium were 5μg / mL of insulin, 5μg / mL of transferrin and 5ng / mL of sodium selenite. SD rat follicles with a diameter of 100-150μm, intact basement membrane, central spherical oocytes and granulosa cells (GCs) around them were selected for scaffold implantation experiments in the decellularized scaffold, thereby constructing an artificial ovarian complex. The implantation density of the follicles was 30 follicles per 1cm 2 Adipose decellularized scaffolds were cultured for 24-72 hours, and the medium was changed every other day during the culture process to obtain artificial ovary complexes.

[0096] Example 3

[0097] The present invention provides a method for preparing a SD rat decellularized scaffold, comprising the following steps:

[0098] 1) Pretreatment of fat and peritoneal tissue: The peritoneal tissue of the abdominal wall around the ovaries of SD rats was taken on a clean bench and recorded as the tissue to be processed. The tissue to be processed was fully washed with PBS to remove impurities. The tissue was sliced ​​into thin slices of about 4 mm × 4 mm × 2 mm with a sterile blade under low-temperature freezing. After slicing, the sterile decellularization process was started.

[0099] Among them, peritoneal tissue is peritoneal tissue of the inner wall of the abdominal cavity close to the level of the ovaries. The cryogenic freezing state is to maintain the fat and peritoneal tissue below 0°C in a solid state. The thickness of the fat and peritoneal tissue is controlled to be about 2 mm. At the same time, ophthalmic forceps and ophthalmic fine shears can be used to remove larger blood vessels and blood clots from fat and peritoneal tissue during the pretreatment process to improve the decellularization effect of subsequent processes.

[0100] 2) Multiple decellularization treatment: Take the fat and peritoneal tissue treated in step 1), first, wash the tissue with ddH2O on a shaker for 30 minutes, repeat 3 times. Each wash needs to be replaced with a new ddH2O washing solution, and the purpose of repeated washing is to remove residual blood, blood clots and lymph as much as possible. Then, wash the tissue with 10% NaCl solution for 2 hours, and then place it on a sterile shaker of ddH2O and PBS for 3 times, each time for 30 minutes. The fat and peritoneal tissue slices are further frozen and thawed three times at -80°C and 37°C, respectively. Then immerse in 4 times the volume of 1% SDS and shake and wash at room temperature for 4 hours, and replace SDS every hour. Wash again with PBS at room temperature for 30 minutes each time to remove the remaining SDS. Finally, incubate the tissue slices with DNase and lipase for 24 hours to obtain a peritoneal decellularized scaffold. Wash with PBS 3 times, each time for 30 minutes. Store at -80°C for later use.

[0101] Wherein, 1% SDS is sodium dodecyl sulfate solution, which is a double distilled water solution of sodium dodecyl sulfate, and the mass volume ratio between the sodium dodecyl sulfate and double distilled water is 1%. The environment is a sterile shaker, the temperature is 37°C, and the stirring speed is controlled to be 100rpm. The concentration of the DNase is 100U / mL. The concentration of the lipase is 5U / mL.

[0102] The present invention also proposes an artificial ovary complex of SD rats, which is obtained by implanting follicles and ovarian stromal cells into a decellularized scaffold. The artificial ovary complex has the function of repairing ovarian damage and reconstructing ovarian function in vitro. Specifically, it includes:

[0103] The decellularized scaffold was pre-soaked in αMEM culture medium for 25 minutes, wherein the α-MEM culture medium contained fetal bovine serum, ITS, follicle stimulating hormone and penicillin-streptomycin mixture. The volume percentage of fetal bovine serum was 10%, the volume percentage of the penicillin-streptomycin mixture was 1%, the working concentration of penicillin in the culture medium was 100U / ml, and the working concentration of streptomycin was 0.1mg / ml. The concentration of follicle stimulating hormone was 0.15mIU / mL. The ITS was composed of insulin, transferrin and sodium selenite, and the concentrations in the culture medium were 5μg / mL insulin, 5μg / mL transferrin and 5ng / mL sodium selenite, respectively. SD rat follicles with a diameter of 100-150μm, intact basement membrane, central spherical oocytes, and granulosa cells (GCs) around them were selected for scaffold implantation experiments in the decellularized scaffold, thereby constructing an artificial ovarian complex. The implantation density of the follicles was 30 follicles per 1cm 2 Peritoneal decellularized scaffolds were cultured for 72 hours, with the medium changed every other day during the culture process, to obtain artificial ovary complexes.

[0104] The above embodiments are only the best examples using SD rats, and are not intended to limit the embodiments of the present invention. In addition to the above embodiments, the present invention has other embodiments. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A decellularized scaffold, characterized in that: The decellularized scaffold is prepared by the following preparation method: 1) taking the ovarian peripheral tissue of a mammal and recording it as the tissue to be processed, removing impurities and slicing the tissue to be processed to obtain tissue slices; 2) Multiple decellularization treatments, including: 2.1) Wash tissue sections with double distilled water and NaCl solution in sequence to separate cells from the surrounding extracellular matrix; 2.2) using an ultra-low temperature refrigerator and an incubator to perform multiple freezing and thawing processes on the tissue sections processed in 2.1); 2.3) washing the tissue sections treated in 2.2) with sodium dodecyl sulfate solution and PBS solution in sequence, so that the nuclei and cytoplasmic components inside the cells are completely released; 2.4) The tissue sections treated in 2.3) are incubated in DNAse and lipase in sequence to remove the nucleic acid and fat components in the tissue sections, thereby preparing acellular scaffolds, which are then washed multiple times with PBS solution and finally frozen for future use.

2. The decellularized scaffold according to claim 1, characterized in that: In the above 1), the size of the tissue slice is 3-5 mm×3-5 mm×1-2 mm.

3. The decellularized scaffold according to claim 1, characterized in that: The 2.1) is specifically: First, wash the tissue sections several times with double distilled water on a shaker; then, wash the tissue with NaCl solution by shaking for several hours; then, wash the tissue sections several times with double distilled water on a shaker; finally, wash the tissue sections several times with PBS solution on a shaker.

4. The decellularized scaffold according to claim 1, characterized in that: The 2.2) is specifically: 2.1) The treated tissue sections were frozen in a -80°C ultra-low temperature refrigerator and thawed in a 37°C incubator, and this process was repeated multiple times.

5. The decellularized scaffold according to claim 1, characterized in that: The ovarian surrounding tissue is one or more of adipose tissue and abdominal wall peritoneal tissue.

6. An artificial ovary complex, characterized in that: The artificial ovary complex is obtained by implanting follicles and ovarian stromal cells into the decellularized scaffold of claim 1, and the artificial ovary complex has the function of repairing ovarian damage and reconstructing ovarian function in vitro.

7. The artificial ovary complex according to claim 6, characterized in that: The preparation process of the artificial ovary complex specifically includes: The decellularized scaffold was pre-soaked in αMEM culture medium for several minutes, and follicles with a diameter of 100-150 μm, intact basement membrane, central spherical oocytes and surrounding granulosa cells were selected for scaffold implantation experiments in the decellularized scaffold to construct an artificial ovarian complex.

8. A method for preparing a decellularized scaffold, characterized in that: The preparation method comprises the following steps: 1) taking the ovarian peripheral tissue of a mammal and recording it as the tissue to be processed, removing impurities and slicing the tissue to be processed to obtain tissue slices; 2) Multiple decellularization treatments, including: 2.1) Wash tissue sections with double distilled water and NaCl solution in sequence to separate cells from the surrounding extracellular matrix; 2.2) using an ultra-low temperature refrigerator and an incubator to perform multiple freezing and thawing processes on the tissue sections processed in 2.1); 2.3) washing the tissue sections treated in 2.2) with sodium dodecyl sulfate solution and PBS solution in sequence, so that the nuclei and cytoplasmic components inside the cells are completely released; 2.4) The tissue sections treated in 2.3) are incubated in DNAse and lipase in sequence to remove the nucleic acid and fat components in the tissue sections, thereby preparing acellular scaffolds, which are then washed multiple times with PBS solution and finally stored at -80°C for future use.

9. The method for preparing a decellularized scaffold according to claim 8, characterized in that: The 2.1) is specifically: First, wash the tissue sections several times with double distilled water on a shaker; then, wash the tissue with NaCl solution by shaking for several hours; then, wash the tissue sections several times with double distilled water on a shaker; finally, wash the tissue sections several times with PBS solution on a shaker.

10. Use of a decellularized scaffold in preparing products for ovarian damage and reconstruction of ovarian function, characterized in that: The ovarian damage includes any one of ovarian damage caused by aging, ovarian damage caused by genetic or immune function defects or environment, iatrogenic ovarian damage or pathological ovarian damage.

Images