Preparation method of thin endometrial repair biological scaffold

Abstract

The invention discloses a preparation method of a biological scaffold for thin endometrial repair, and belongs to the technical field of biological medicines.The preparation method comprises the steps that S1, blood clots of umbilical cord tissue donated by a healthy patient are removed, and then the umbilical cord tissue is cut into small pieces; putting the umbilical cord tissue into a culture dish until the umbilical cord tissue blocks are adhered to the wall; step S2, culturing the primary uMSCs in a culture medium; step S3, when the uMSCs in the culture medium reach 80-90% fusion, carrying out subculture; step S4, dissolving freeze-dried fetal calfskin type I collagen in an acetic acid solution, and fully swelling to obtain a collagen solution; step S5, neutralizing the collagen solution; step S6, digesting the uMSCs with 0.25% pancreatin, centrifuging, and then collecting cells; and step S7, mixing the uMSCs with a collagen solution to ensure that the concentration of the uMSCs is not less than 1 * 10 < 6 > / mL, and gelatinizing. The collagen hydrogel is combined with the uMSCs, so that dryness of the uMSCs is expected to be kept, the duration time of the uMSCs in a uterine cavity is prolonged, and the endometrial regeneration and fertility capabilities are enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for preparing a thin endometrial repair biological scaffold. Background Art

[0002] The successful implantation and development of the embryo depends on good embryo quality and acceptable endometrium. Due to the increase in intrauterine surgeries such as dilation and curettage, the incidence of endometrial receptivity defects has increased year by year. Endometrial thickness is an important factor affecting endometrial receptivity. Insufficient thickness of the endometrial lining makes it difficult to maintain a healthy pregnancy. A variety of strategies have been developed clinically to improve endometrial thickness, including hormone replacement therapy (HRT), aspirin, sildenafil, etc. However, these treatments may cause serious side effects, such as bleeding or increased risk of tumors.

[0003] Uterine homeostasis and self-regeneration are regulated by endometrial stem cells located in the basement membrane of the uterine cavity. Thin endometrium is mainly caused by insufficient cell proliferation and is associated with damage or deficiency of endometrial stem cells. Human umbilical cord mesenchymal stem cells (uMSCs) are a type of stem cell with low immunogenicity and sufficient donors that do not require secondary surgery. They are considered to be promising seed cells for the treatment of thin endometrium. By transplanting uMSCs, the structure and function of thin endometrium were significantly restored. Although uMSCs-based therapy has achieved initial promising results in improving the regeneration of thin endometrium, the low survival or homing rate of transplanted cells limits the efficiency of treatment.

[0004] Tissue engineering aims to restore damaged tissues by combining cells and biomaterial scaffolds. It is an ideal clinical solution for enhancing stem cell therapy. By accelerating cell survival and attachment, the lineage-specific differentiation of stem cells encapsulated in scaffolds can be significantly enhanced. Collagen is a protein widely present in the extracellular matrix of mammals. It has a wide range of sources and good biocompatibility and biodegradability. It is widely used in cell delivery to damaged tissues. The network structure formed by cross-linking can anchor and support cell growth and promote cell retention in extreme environments in the body. Collagen hydrogels also have the ability to prevent stem cell transformation induced by monolayer expansion. Stem cells seeded on collagen patches have been used to improve damaged endometrium caused by curettage and / or endometritis. However, these collagen patches cannot adapt to the irregular shape of the uterine cavity, and the implantation procedure is more complicated than the minimally invasive injection of hydrogels, which limits clinical application; in addition, the improvement of fertility by collagen patch transplantation is still unsatisfactory. Summary of the invention

[0005] To solve the above technical problems, the present application combines injectable collagen hydrogel with uMSCs for thin endometrium. The collagen hydrogel combined with uMSCs is expected to maintain the stemness of uMSCs and prolong their duration in the uterine cavity, thereby enhancing endometrial regeneration and fertility.

[0006] To achieve the above objectives, the present application aims to propose a method for preparing a thin endometrial repair biological scaffold, the preparation method comprising:

[0007] Step S1, removing blood clots from umbilical cord tissue donated by healthy patients and cutting the umbilical cord tissue into small pieces; placing the umbilical cord tissue in a culture dish until the umbilical cord tissue pieces adhere to the wall;

[0008] Step S2, culturing primary uMSCs in culture medium;

[0009] Step S3, when the uMSCs in the culture medium reach 80-90% confluence, subculture;

[0010] Step S4, dissolving freeze-dried fetal bovine skin type I collagen in an acetic acid solution, and fully swelling the solution to obtain a collagen solution;

[0011] Step S5, neutralizing the collagen solution;

[0012] Step S6, uMSCs were digested with 0.25% trypsin and centrifuged to collect the cells;

[0013] Step S7: Mix uMSCs with collagen solution, and the concentration of uMSCs is not less than 1×10 6 / mL and gelled.

[0014] Furthermore, the culture medium in step S2 includes 9-12% fetal bovine serum and 0.8-2% penicillin-streptomycin solution.

[0015] Furthermore, in step S3, the uMSCs are cultured in an incubator at 37° C., 94-98% humidity, and 4-6% CO 2 concentration.

[0016] Furthermore, the concentration of acetic acid in step S4 is 0.4-0.6M.

[0017] Furthermore, the swelling temperature in step S4 is 3-5°C.

[0018] Furthermore, in step S5, the collagen solution is neutralized to pH 7 using 0.8-1.2 M NaOH at 3-5°C;

[0019] The collagen solution is neutral, and the final concentration is 10-14 mg / mL.

[0020] Furthermore, in step S6, the uMSCs are centrifuged at 1000 rpm for 5 to 6 minutes.

[0021] Furthermore, the uMSCs are mixed with the collagen solution and then gelled at 37° C. for 10 to 12 minutes.

[0022] Compared with the prior art, the advantages and effects of this application are as follows:

[0023] 1. The present invention utilizes injectable collagen hydrogel in combination with uMSCs for thin endometrial regeneration. The collagen hydrogel combined with uMSCs is expected to maintain the stemness of uMSCs and prolong their duration in the uterine cavity, thereby enhancing endometrial regeneration and fertility.

[0024] 2. The present invention adds uMSCs to collagen hydrogel for culture, which can maintain the stemness of uMSCs. Stem cell-based treatments are always expanded in vitro to obtain sufficient cells, and maintaining the stemness of uMSCs plays a vital role in its therapeutic effect.

[0025] 3. The present invention utilizes injectable collagen hydrogel and uMSCs to prepare a thin endometrial repair biological scaffold. First, the collagen hydrogel can be naturally degraded in the body. When the collagen hydrogel wraps the uMSCs, they will not be quickly discharged from the uterine cavity, which prolongs their duration in the uterine cavity and retains the uMSCs in the uterus to repair damage.

[0026] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a detailed description of the preferred embodiments of the present application in conjunction with the accompanying drawings as follows.

[0027] Based on the detailed description of the specific embodiments of the present application in combination with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0029] in:

[0030] Figure 1 Characterization of collagen hydrogel and its effect on uMSCs survival rate and stemness:

[0031] 1A is a diagram showing the process of neutral collagen solution forming collagen hydrogel;

[0032] 1B is the rheological properties diagram of collagen hydrogel under 1%-100% strain;

[0033] 1C is the rheological properties diagram of collagen hydrogel at a frequency of 0.1-1 Hz;

[0034] 1D is a SEM showing the microstructure of collagen (scale bars: 50 μm and 25 μm);

[0035] 1E is the proliferation statistics of uMSCs in two-dimensional culture plates, collagen patches, and collagen hydrogels;

[0036] 1F is a statistical graph of the survival rate of uMSCs encapsulated in collagen hydrogels at 24 h (scale bars: 5 mm and 200 μm);

[0037] 1G is a statistical graph of the survival rate of uMSCs encapsulated in collagen hydrogel at 72 hours (scale bars: 5 mm and 200 μm);

[0038] 1H is the stem gene expression graph after 24 hours;

[0039] 1I is the expression graph of stem genes after 72 hours;

[0040] Figure 2 This is a tracer image of collagen hydrogel / uMSCs implanted in the uterus:

[0041] 2A is the HE staining image of collagen hydrogel / uMSCs in vivo after transplantation;

[0042] 2B is the transplantation of an estrus cycle diagram;

[0043] 2C is a diagram of transplantation of two estrus cycles;

[0044] 2D is the distribution diagram of uMSCs in the rat uterus after one estrus cycle after implantation;

[0045] 2E is a quantitative analysis of the content of uMSCs after one estrus cycle;

[0046] 2F is the distribution diagram of uMSCs in the uterus of rats in two estrus cycles after different treatments;

[0047] 2G is a statistical analysis of the uMSCs content after two estrus cycles;

[0048] Figure 3This is a morphological evaluation diagram of the regenerated uterus in rats:

[0049] 3A is a diagram showing the steps for establishing a rat thin endometrium model.

[0050] 3B is the HE staining image reflecting the endometrial morphology after different treatments.

[0051] 3C is a comparison chart of the endometrial thickness in each group.

[0052] 3D is a comparison chart of the number of glands in each group;

[0053] Figure 4 Figure 1: Immunohistochemical evaluation of the regenerating uterus.

[0054] 4A is the immunohistochemical staining of CD34, vimentin, α-SMA, and αvβ3;

[0055] 4B is a graph showing the positive area of ​​quantification of CD34 immunohistochemical staining

[0056] 4C is a graph showing the positive area of ​​quantitative vimentin immunohistochemical staining;

[0057] 4D is the positive area graph of quantitative a-SMA immunohistochemical staining;

[0058] 4E is a graph of the H-score for αvβ3 expression. Specific embodiments

[0059] To make the purpose, technical scheme and advantages of the embodiment of the present application clearer, the technical scheme in the embodiment of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present application. Obviously, the described embodiment is a part of the embodiment of the present application, rather than all of the embodiments. In the following description, specific details such as specific configuration and components are provided only to help fully understand the embodiments of the present application. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, for clarity and brevity, the description of known functions and structures is omitted in the embodiment.

[0060] It should be understood that the references to "one embodiment" or "this embodiment" throughout the specification mean that the specific features, structures, or characteristics associated with the embodiment are included in at least one embodiment of the present application. Therefore, the references to "one embodiment" or "this embodiment" appearing throughout the specification do not necessarily refer to the same embodiment. In addition, these specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0061] In addition, the present application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplicity and clarity, and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0062] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that there can be two relationships. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0063] The term "at least one" in this article is merely a description of the association relationship of associated objects, indicating that there may be three relationships. For example, at least one of A and B can mean: A exists alone, A and B exist at the same time, and B exists alone.

[0064] It should also be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusions.

[0065] Example 1

[0066] This embodiment discloses a method for preparing a thin endometrial repair biological scaffold, the preparation method comprising:

[0067] Step S1, removing blood clots from umbilical cord tissue donated by healthy patients and cutting the umbilical cord tissue into small pieces; placing the umbilical cord tissue in a culture dish until the umbilical cord tissue pieces adhere to the wall;

[0068] Step S2, culturing primary uMSCs in culture medium;

[0069] Step S3, when the uMSCs in the culture medium reach 80-90% confluence, subculture;

[0070] Step S4, dissolving freeze-dried fetal bovine skin type I collagen in an acetic acid solution, and fully swelling the solution to obtain a collagen solution;

[0071] Step S5, neutralizing the collagen solution;

[0072] Step S6, uMSCs were digested with 0.25% trypsin and centrifuged to collect the cells;

[0073] Step S7: Mix uMSCs with collagen solution, and the concentration of uMSCs is not less than 1×10 6 / mL and gelled.

[0074] Furthermore, the culture medium in step S2 includes 10% fetal bovine serum and 1% penicillin-streptomycin solution.

[0075] Furthermore, in step S3, the uMSCs are cultured in an incubator at 37° C., 95% humidity and 5% CO 2 concentration.

[0076] Furthermore, the concentration of acetic acid in step S4 is 0.5M.

[0077] Furthermore, the swelling temperature in step S4 is 4°C.

[0078] Furthermore, in step S5, the collagen solution is neutralized to pH 7 at 4° C. using 1 M NaOH;

[0079] The collagen solution is neutral, and the final concentration is 12 mg / mL.

[0080] Furthermore, in step S6, the uMSCs are centrifuged at 1000 rpm for 5 minutes.

[0081] Furthermore, the uMSCs and the collagen solution are mixed and gelled at 37° C. for 10 minutes.

[0082] This embodiment utilizes injectable collagen hydrogel in combination with uMSCs for thin endometrial regeneration. The combination of collagen hydrogel and uMSCs is expected to maintain the stemness of uMSCs and prolong their duration in the uterine cavity, thereby enhancing endometrial regeneration and fertility.

[0083] Example 2

[0084] This embodiment discloses a method for preparing a thin endometrial repair biological scaffold, please refer to Figure 1 As shown, further introduction is given based on the above embodiments.

[0085] First, please refer to Figure 1 As shown in A, collagen hydrogel is a temperature- and pH-sensitive hydrogel, the solution of which is transparent and injectable under acidic and low-temperature conditions, and gels under neutral conditions by incubation at 37°C for 10 minutes, which makes the collagen hydrogel minimally invasive and can reside in the uterine cavity.

[0086] For further information, please refer to Figure 1 B and 1C, Figure 1B represents the rheological properties of collagen hydrogel under 1%-100% strain. The results show that the intersection of G' and G" of collagen hydrogel confirms the formation of hydrogel; Figure 1 C represents the rheological properties of collagen hydrogel at a frequency of 0.1-1 Hz. The results show that G' is always higher than G", indicating that collagen hydrogel forms a stable viscoelastic solid.

[0087] For further information, please refer to Figure 1 As shown in Figure D, the microstructure of the collagen hydrogel was observed by SEM, and the collagen hydrogel showed a porous structure with a pore size of 50-150μm. Hydrogels with a pore size of about 50μm are common, which is conducive to the exchange of metabolites and nutrients between cells and the outside world.

[0088] This example describes that the collagen hydrogel is transparent and injectable under acidic and low temperature conditions, and gels under neutral conditions by incubation at 37°C for 10 minutes, which makes the collagen hydrogel minimally invasive and can reside in the uterine cavity, which reduces the abnormal mechanical load caused by tissue expansion and improves adaptive remodeling.

[0089] Example 3

[0090] This embodiment discloses a method for preparing a thin endometrial repair biological scaffold, please refer to Figure 1 As shown, further introduction is given based on the above embodiments.

[0091] Please refer to Figure 1 As shown in E, the results of CCK-8 showed that the cells cultured on the collagen patch (Collagen+uMSCs group) and in the collagen hydrogel (Collagen@uMSCs group) exhibited good biocompatibility.

[0092] For further information, please refer to Figure 1 F and Figure 1 As shown in G, the overall data showed that uMSCs were able to survive and exhibit proliferation activity in collagen patches and collagen hydrogels, with no significant differences among cultures for 24, 48, and 72 hours.

[0093] Cell viability assay showed that a small number of dead cells (red) and a large number of live cells (green) were observed at 24 and 72 h, indicating that culture in collagen hydrogels maintained the survival rate of uMSCs.

[0094] Furthermore, stem cell-based therapies always require in vitro expansion to obtain sufficient cells, a process that has many adverse effects on the stemness of stem cells. Therefore, maintaining the stemness of MSCs plays a vital role in their therapeutic effects.

[0095] Please refer to Figure 1 H and Figure 1As shown in Figure 1, culturing stem cells in collagen hydrogels can prevent MSCs transformation caused by monolayer expansion. qRT-PCR was performed to detect the expression of stemness genes (SOX2, Oct-4, Nanog) in uMSCs cultured in collagen hydrogels.

[0096] The expression levels of SOX2, Oct-4, and Nanog in uMSCs cultured in 3D collagen hydrogels were upregulated compared with those cultured on flat plates and collagen patches. Figure 1 H shows that the stemness genes cultured in collagen hydrogel increased by 6.8%, 75.4%, and 30.7% after 24 hours; Figure 1 I shows that the stemness of genes cultured in collagen hydrogels increased by 31.4%, 45.8% and 45.0% respectively after 72 hours.

[0097] The data of this example show that the stemness of uMSCs can be maintained by culturing them in collagen hydrogel.

[0098] Example 4

[0099] This embodiment discloses a method for preparing a thin endometrial repair biological scaffold, please refer to Figure 2 As shown, further introduction is given based on the above embodiments.

[0100] Please refer to Figure 2 As shown in A, to track the degradation of transplanted collagen hydrogel / uMSCs in vivo, the uterus was stained with HE after transplantation; Figure 2 B shows the status of one estrus cycle after transplantation and it was observed that collagen hydrogel / uMSCs were slowly degraded in the uterine cavity; Figure 2 C shows the state two estrous cycles after transplantation, and the collagen hydrogel in the uterine cavity disappears; these results indicate that the collagen hydrogel can be naturally degraded in vivo.

[0101] Furthermore, the retention of uMSCs in tissue defects is crucial for damage repair, please refer to Figure 2 As shown in D-2G, no fluorescence signals of uMSCs were detected in the sham group, model group, and collagen group. In one and two estrus cycles after different treatments, as shown by the quantification of cell numbers in the uMSCs and Collagen@uMSCs groups, the retention of uMSCs in transplantation was significantly higher in Collagen@uMSCs than in uMSCs alone (p<0.05). This may be due to the fact that uMSCs were encapsulated in collagen hydrogel to avoid rapid expulsion from the uterine cavity.

[0102] The experimental data shown in this example indicate that collagen hydrogel can be naturally degraded in vivo. When uMSCs are encapsulated in collagen hydrogel, they will not be rapidly discharged from the uterine cavity. The uMSCs remain in the uterus and can be damaged and repaired.

[0103] Example 5

[0104] This embodiment discloses a method for preparing a thin endometrial repair biological scaffold, please refer to Figure 3 As shown, further introduction is given based on the above embodiments.

[0105] The number of glands and thickness of the endometrium are crucial for successful embryo reception and development. Figure 3 As shown in A, a thin endometrium rat model was established by intrauterine injection of 95% alcohol; Figure 3 As shown in B, under the intervention of 95% alcohol, the endometrial thickness and gland number of the model group were significantly reduced compared with the sham operation group, indicating that the thin endometrium model was successfully established in rats.

[0106] For further information, please refer to Figure 3 As shown in C and 3D, the average endometrial thickness and gland number in the sham operation group were 478.6±40.0μm and 14.2±2.8, respectively, while those in the model group dropped to 129.2±56.5μm and 1.2±1.1, respectively. Injection of collagen hydrogel did not improve the condition of the endometrium.

[0107] However, the thin endometrium was partially rescued after treatment with uMSCs or Collagen@uMSCs, with the thickness increased to 331.8±33.3μm and 408.0±19.6μm, respectively, and the number of glands increased to 7.4±1.8 and 12.2±2.2, respectively, which was consistent with the endometrial thickness. Specifically, the endometrial thickness and gland number in the Collagen@uMSCsuMSCs group were 0.23 times and 0.64 times that of the sham group, respectively, which were close to those in the sham group (0.85 times and 0.86 times).

[0108] These results in this example highlight the beneficial effects of the combination of collagen and uMSCs in the treatment of thin endometrium.

[0109] Example 6

[0110] This embodiment discloses a method for preparing a thin endometrial repair biological scaffold, please refer to Figure 4 As shown, further introduction is given based on the above embodiments.

[0111] The angiogenesis process is required for uterine tissue regeneration to replenish oxygen and nutrients, and CD34, a vascular endothelial cell marker, is used to indicate the blood vessel density in the regenerating endometrium.

[0112] Please combine Figure 4 A. Figure 4 As shown in B, immunohistochemical staining for CD34 was performed to examine whether the combined transplantation of collagen and uMSCs affects angiogenesis in the regenerated uterus. Compared with the model group (3.8% ± 1.3%), the CD34-positive staining area in the uMSCs (13.8% ± 2.7%) and Collagen@uMSCs groups (20.8% ± 1.8%) increased significantly, by 263.2% and 447.4%, respectively. However, the intervention of collagen hydrogel alone did not change significantly (5.3% ± 2.6%) (p>0.05); by the combined intervention of collagen hydrogel and uMSCs, the CD34-positive staining area was close to that of the sham group (25.3% ± 2.6%), which was 0.51 times higher than that of uMSCs alone.

[0113] Furthermore, vimentin, a protein expressed in the cytoplasm of endometrial stromal cells, was used to detect the proliferation of endometrial stromal cells in the regenerating uterus by immunohistochemical staining.

[0114] Please combine Figure 4 A. Figure 4 As shown in C, the percentage of vimentin-positive area in the Collagen@uMSCs group (20.5% ± 4.0%) was significantly higher than that in the model group (3.5% ± 1.5%), collagen group (3.7% ± 1.8%), and uMSCs group (14.4% ± 3.7%), and compared with the sham group (27.1% ± 3.3%) (p < 0.05). The results showed that uMSCs can promote the proliferation of endometrial stromal cells, and this effect can be increased to 142.4% when combined with collagen.

[0115] Furthermore, in late pregnancy, the uterine muscle layer is too thin and adverse events such as uterine rupture may occur, which may result in maternal and fetal death.

[0116] To evaluate the regeneration of the myometrium, the expression of a-SMA in the rat myometrium was detected. Figure 4 A. Figure 4 As shown in D, the results showed that after 95% alcohol treatment, the rat myometrium became thinner and negatively stained for α-SMA (43.5% ± 2.5%) compared with the sham group. Although the myometrium thickness and the expression of a-SMA in uMSCs (32.3% ± 2.5%) and collagen@uMSCs (38.5% ± 2.8%) in the treatment group were significantly upregulated compared with the model group (26.6% ± 1.6%), there was no significant difference in the collagen hydrogel injection alone (28.4% ± 1.3%). In addition, they were statistically significant in the collagen@uMSCs group compared with the uMSCs transplantation alone group (p < 0.01).

[0117] Furthermore, these results emphasize that the combination of collagen hydrogel and uMSCs is more beneficial for maintaining late pregnancy, and endometrial receptivity is one of the prerequisites for successful pregnancy.

[0118] Please combine Figure 4 A. Figure 4 As shown in E, integrin αvβ3 is mainly expressed in the endometrial luminal epithelium and glandular epithelium and is a well-known marker of endometrial receptivity. Immunohistochemical positive staining and H scores decreased sharply in the administration of 95% ethanol. There was a slight difference in the scores between the model group (111.7±10.6) and the collagen group (122.8±21.1), indicating that the collagen hydrogel had little effect on the restoration of endometrial receptivity. However, the scores of the uMSCs (171.6±15.3) and collagen@uMSCs (232.4±18.7) groups were only reduced by 31.0% and 6.6% compared with the sham group (248.8±22.0).

[0119] The above description is only the preferred embodiment of the present invention, which does not limit the protection scope of the present invention. For those skilled in the art, the present invention can be modified and varied in various ways. Within the spirit and principle of the present invention, any change, modification, replacement, integration and parameter change of these embodiments by conventional substitution or capable of achieving the same function without departing from the principle and spirit of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a thin endometrial repair biological scaffold, characterized in that: The preparation method comprises: Step S1, removing blood clots from umbilical cord tissue donated by healthy patients and cutting the umbilical cord tissue into small pieces; placing the umbilical cord tissue in a culture dish until the umbilical cord tissue pieces adhere to the wall; Step S2, culturing primary uMSCs in culture medium; Step S3, when the uMSCs in the culture medium reach 80-90% confluence, subculture; Step S4, dissolving freeze-dried fetal bovine skin type I collagen in an acetic acid solution, and fully swelling the solution to obtain a collagen solution; Step S5, neutralizing the collagen solution; Step S6, uMSCs were digested with 0.25% trypsin and centrifuged to collect the cells; Step S7: mixing uMSCs with collagen solution, wherein the concentration of uMSCs is not less than 1×10 6 / mL and gelled.

2. The method for preparing a thin endometrial repair biological scaffold according to claim 1, characterized in that: The culture medium in step S2 includes 9-12% fetal bovine serum and 0.8-2% penicillin-streptomycin solution.

3. The method for preparing a thin endometrial repair biological scaffold according to claim 1, characterized in that: In step S3, the uMSCs are cultured in an incubator at 37°C, 94-98% humidity and 4-6% CO2 concentration.

4. The method for preparing a thin endometrial repair biological scaffold according to claim 1, characterized in that: The concentration of acetic acid in step S4 is 0.4-0.6M.

5. A method for preparing a thin endometrial repair biological scaffold according to claim 1 or 4, characterized in that: The swelling temperature in step S4 is 3-5°C.

6. The method for preparing a thin endometrial repair biological scaffold according to claim 1, characterized in that: In step S5, the collagen solution is neutralized to pH 7 with 0.8-1.2 M NaOH at 3-5°C; The collagen solution is neutral, and the final concentration is 10-14 mg / mL.

7. The method for preparing a thin endometrial repair biological scaffold according to claim 1, characterized in that: In step S6, the uMSCs are centrifuged at 1000 rpm for 5 to 6 minutes.

8. The method for preparing a thin endometrial repair biological scaffold according to claim 1, characterized in that: The uMSCs and the collagen solution are mixed and gelled at 37° C. for 10 to 12 minutes.

Images