Preparation method of human-derived mesenchymal stem cell membrane

Abstract

The invention belongs to the technical field of organic compounding, and the prepared stem cell membrane can completely retain cell surface protein, extracellular matrix and intercellular tight connection, and is beneficial to enhancing stem cell activity and maintaining local effective cell concentration. Through laminated construction of the three-dimensional bionic tissue, animal in-vivo experiment results show that regeneration of bone tissues in a defect area can be remarkably promoted, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of inorganic and organic composites, and particularly relates to the application of human mesenchymal stem cell sheets in craniofacial bone defects. Background Art

[0002] Bone defects are often caused by congenital deformities, bone injuries, bone tumors, etc. According to a report released by the "International Osteoporosis Foundation", bone injuries affect more than 200,000 people globally every year, resulting in a fracture every 3 seconds. Solving substantial bone defects and promoting and restoring the regeneration of fresh bone tissue have become obstacles that urgently need to be overcome in global repair and reconstruction.

[0003] Bone defect repair and functional reconstruction, especially the regeneration and reconstruction of large-area bone defect tissues caused by infection, trauma, congenital bone diseases, and bone tumors, have become urgent problems to be solved at this stage. People urgently hope that bone defect repair can develop from simple instrument fixation to tissue reconstruction, and from functional replacement to permanent regenerative repair.

[0004] The treatment methods for bone defects include autologous bone transplantation, allogeneic bone transplantation, and reconstruction with biomaterial implants. Autografts have good biocompatibility, but there are limitations in the amount of donor bone and shaping, so their application is limited in the repair of large-scale tissue defects. In addition, a second surgical area needs to be opened during the operation, resulting in a wide range of trauma, a relatively slow recovery speed of the patient after the operation, and the patient may face problems such as hernia, bleeding, nerve injury, tissue necrosis, etc. In severe cases, systemic infection and insufficient blood circulation will ultimately lead to the failure of bone replacement surgery. Allogeneic bone transplantation is mainly based on allogeneic and xenogeneic grafts. The grafts are taken from the bones of others or animals and are used for bone defect repair after being treated by some special means to remove antigens. The main advantages of this technology are that the amount of donor bone is relatively sufficient, and a second surgical area does not need to be opened during the operation, causing less trauma to the patient. However, although allogeneic bone has undergone corresponding treatment, it still has certain immunogenicity. After transplantation, there are still risks of tissue rejection, disease transmission, and ethical issues, so its clinical application is also very limited. In recent years, the application scope of biomaterials in clinical practice has been continuously broadened, and the treatment effect of bone tissue defect repair has also been significantly improved. However, at present, no material has been found that meets all the requirements for bone defect repair. Therefore, most bone substitute materials still need to be further improved in terms of biocompatibility, degradation rate, and mechanical strength.

[0005] Stem cell technology is one of the most cutting-edge scientific and technological fields in the world today. The clinical application of stem cell transplantation technology will bring revolutionary changes to traditional medical methods. By using stem cell-related technologies, through stem cell transplantation, differentiation, and tissue regeneration, it is expected to promote the repair of body trauma and achieve effective regeneration of bone tissue. The application of stem cells in regenerative medicine depends on accurately reaching the lesion site to function, so the method of cell transplantation is crucial. Traditional cell injection technology has limited tissue regeneration effects due to the large cell fluidity, poor mechanical properties, and low retention rate. In 1993, Japanese scholars such as Terou Okano et al. first proposed the cell sheet technology. This technology does not require enzymatic digestion and can form a dense cell sheet tissue by stimulating the secretion of the extracellular matrix. Applying the cell sheet technology can completely retain cell surface proteins, extracellular matrix, and cell-cell tight junctions, which is beneficial to enhancing the activity of stem cells, maintaining the local effective cell concentration, and constructing a three-dimensional biomimetic tissue through cell sheet stacking to effectively repair tissue defects and improve organ function. Currently, this technology has entered the clinical application or experimental stage in multiple fields such as the heart, cornea, esophagus, cartilage, and periodontal in Japan. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a method for preparing a human mesenchymal stem cell sheet:

[0007] Isolation, culture, and identification of human mesenchymal stem cells

[0008] Human mesenchymal stem cells can be derived from tissues such as bone marrow, adipose tissue, umbilical cord, periodontal ligament, and dental pulp. After cell isolation and culture, their proliferation ability, surface markers, and multi-directional differentiation ability are identified.

[0009] Preparation of stem cell sheets

[0010] The seeding density of in vitro-cultured human mesenchymal stem cells can be 1×105 - 2×105 cells / cm2. They are seeded into a conventional culture dish or culture plate, and cultured in a basic medium for 24 h, and then changed to a special medium, with the medium changed once every 3 days.

[0011] The basic medium includes: α-MEM, 10 v / v% fetal bovine serum (FBS), 1 v / v% penicillin-streptomycin (PS). The special medium includes: basic medium, 1 v / v% ITS, 0.04 μg / mL dexamethasone, 15 mM HEPES, 50 μg / mL vitamin C.

[0012] Obtaining of stem cell sheets

[0013] After culturing in the special medium for 24 h - 7 days, gently and repeatedly pipette the edge of the culture dish with a pipette gun to detach the cells at the edge from the bottom of the culture dish, and gradually detach the whole piece to form a complete cell sheet.

[0014] Performance Detection of Stem Cell Membrane Sheets

[0015] The prepared single-layer stem cell membrane sheets are intact without damage, with a thickness of 10-100 microns and a cell density of 3×105 to 2×106 / cm2. The extracellular matrix of the membrane sheets is rich in fibronectin and integrin-β1, and can secrete one or more of a variety of cytokines, such as BMP-2, IL-6, TGF-β, and VEGF-A.

[0016] Lamination of Stem Cell Membrane Sheets

[0017] For the prepared single-layer stem cell membrane sheets, remove the culture medium and lay them flat on the bottom of the culture dish. Incubate at 37 °C and 5% CO2 for 20 minutes (the first-layer cell membrane sheet). Use a pipette to slowly place the second-layer cell membrane sheet that has been completely peeled off and contains a small amount of culture medium above the first-layer cell membrane sheet, making it fully spread and cover the first-layer cell membrane sheet. Then remove the culture medium again and incubate at 37 °C and 5% CO2 for 20 minutes. Place the third-layer cell membrane sheet above the second-layer cell membrane sheet in the same way, remove the culture medium again, and incubate at 37 °C and 5% CO2 for 20 minutes.

[0018] Transplantation of Laminated Cell Membrane Sheets

[0019] For the cell membrane sheets completed with three-layer stacking, add an appropriate amount of basal medium, and use a pipette gun to blow them up completely from the bottom. Then transplant them to the bone defect area exposed by the surgery. The size and shape of the membrane sheets can be trimmed to adapt to the scope of the bone defect.

[0020] Compared with the existing technologies, the beneficial effects of the present invention are as follows:

[0021] (1) The stem cell membrane sheets prepared by the present invention can completely retain cell surface proteins, extracellular matrix, and tight cell junctions, which is beneficial to enhancing stem cell activity and maintaining the local effective cell concentration.

[0022] (2) The stem cell membrane sheets prepared by the present invention can secrete cytokines better, which is more conducive to the regulation of the local microenvironment and effectively promotes tissue regeneration.

[0023] (3) The stem cell membrane sheets prepared by the present invention construct three-dimensional biomimetic tissues through lamination. The results of in vivo animal experiments show that they can significantly promote the regeneration of bone tissues in the defect area.

[0024] (4) The preparation method provided by the present invention uses a conventional cell culture dish or culture plate to prepare stem cell membrane sheets, with low cost and good repeatability, which is beneficial to the popularization and use of this technology. Description of the Drawings

[0025] Figure 1Results of micro CT for animal experiments. Detailed implementation manners

[0026] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The specific implementation of the present invention will be described in detail below in conjunction with specific embodiments.

[0027] Example 1

[0028] Isolation, culture and identification of bone marrow mesenchymal stem cells

[0029] Isolation and culture of bone marrow mesenchymal stem cells

[0030] The cancellous bone tissue of the alveolar bone (from the outpatient department of oral and maxillofacial surgery in the Stomatological Center of China-Japan Friendship Hospital, approved by the Ethics Committee of China-Japan Friendship Hospital) was washed with PBS and then placed in a pre-cooled α-MEM medium containing 2 v / v% PS. After centrifugation at 1000 rpm for 15 minutes at low temperature, the supernatant was discarded, and α-MEM cell medium was added. The cells were suspended into a single-cell suspension, and the cells were inoculated into 6-well plates at a density of 1x105 cells / well. They were cultured in a 37 °C, 5% CO2 cell incubator, and the medium was changed every 3 days. When the cells grew to 80% confluence, they were digested with 0.1% trypsin / 0.1% EDTA solution and subcultured at a ratio of 1:3.

[0031] Detection of the proliferation ability of bone marrow mesenchymal stem cells

[0032] Here, the CCK-8 method is taken as an example to illustrate the detection of the proliferation ability of bone marrow mesenchymal stem cells. The cells in the logarithmic growth phase were digested with 0.1% trypsin / 0.1% EDTA solution, the cells were resuspended and counted, and then inoculated into 96-well plates at a density of 3×103 cells / well, with 150 μl of medium in each well. The culture plates were placed in a 37 °C, 5% CO2 cell incubator and cultured for 7 days. At 0, 1, 3, 5, and 7 days, 15 μl of CCK-8 solution was added to each well, incubated at 37 °C for 2 hours, and the absorbance (OD 450nm) value at 450 nm was measured with an enzyme-labeled instrument. A growth curve was plotted, and the results showed that the number of cells increased with the extension of time, and entered the logarithmic growth phase on the 3rd day.

[0033] Identification of surface markers of bone marrow mesenchymal stem cells

[0034] Here, flow cytometry was used as an example to illustrate the identification of surface markers of bone marrow mesenchymal stem cells. Cells in the logarithmic growth phase were digested with 0.1% trypsin / 0.1% EDTA solution and resuspended. The surface marker proteins of the cells were stained according to the instructions of the purchased reagents, and detected by flow cytometry. Among them, Vimentin, Nestin, CD105, CD90, CD73, and CD146 were all positively expressed, while CK-19 and CD34 were negatively expressed. Detection of the multi-directional differentiation ability of bone marrow mesenchymal stem cells

[0035] Cells in the logarithmic growth phase were digested with 0.1% trypsin / 0.1% EDTA solution, counted after resuspension, and inoculated into 24-well plates at a density of 2×105 cells / well, with 500 μl of medium in each well. The culture plates were placed in a 37 °C, 5% CO2 cell incubator. When the cells reached 80% confluence, the medium was replaced with the purchased osteogenic, adipogenic, and chondrogenic induction differentiation media, and induced culture was carried out according to the protocol in the reagent instructions, with the medium changed every 2 - 3 days. After 21 days of induction, alizarin red, oil red O, and alcian blue staining were used to detect the osteogenic, adipogenic, and chondrogenic induction differentiation results. Alizarin red staining showed deeply stained red mineralized nodules, oil red O staining showed the formation of red round lipid droplets inside the cells, and alcian blue staining showed the formation of deeply stained blue mucopolysaccharides.

[0036] Example 2

[0037] Preparation and lamination of bone marrow mesenchymal stem cell sheets

[0038] Preparation of bone marrow mesenchymal stem cell sheets

[0039] Bone marrow mesenchymal stem cells cultured in vitro were inoculated into 24-well culture plates at 1×105. After routine culture in the basal medium for 24 h, the medium was replaced with a special medium, and the medium was changed once every 3 days.

[0040] Obtaining of stem cell sheets

[0041] After culturing in the special medium for 7 days, the edge of the culture dish was gently and repeatedly pipetted to detach the cells at the edge from the bottom of the culture dish, and gradually the whole sheet detached to form a complete cell sheet.

[0042] Performance detection of stem cell sheets

[0043] The prepared monolayer stem cell sheets were intact without damage. Their thickness was measured to be 30 μm, and the cell density was 5×105 / cm2. Fibronectin and Integrin-β1 could be detected in the extracellular matrix of the sheets, and various cytokines such as BMP-2, IL-6, TGF-β, and VEGF-A could be detected.

[0044] Live and dead cell staining: The obtained monolayer stem cell membrane sheet was stained using a Calcein-AM / PI double staining kit, and more than 99% of the cells were live cells.

[0045] Laser confocal detection

[0046] Detection of cell membrane sheet secreted factors in the culture supernatant

[0047] Observation of extracellular matrix in the cell membrane sheet by tissue section

[0048] Stacking of stem cell membrane sheets

[0049] The obtained monolayer stem cell membrane sheet had the culture medium removed and was left standing for 20 minutes at 37 °C and 5% CO2 (the first layer of cell membrane sheet). Using a pipette, the second layer of the cell membrane sheet that had been completely peeled off and contained a small amount of culture medium was slowly placed above the first layer of cell membrane sheet, allowing it to fully spread and cover the first layer of cell membrane sheet. The culture medium was removed again and the sheet was left standing for 20 minutes at 37 °C and 5% CO2. The third layer of cell membrane sheet was placed above the second layer of cell membrane sheet in the same way, and the culture medium was removed again and the sheet was left standing for 20 minutes at 37 °C and 5% CO2. For the cell membrane sheet completed with three-layer stacking, an appropriate amount of basal medium was added, and it was completely blown up from the bottom using a pipette gun and placed in reserve at 37 °C and 5% CO2.

[0050] Example 3

[0051] Preparation of a rat calvarial bone defect model

[0052] For 8-week-old male SD rats, under isoflurane inhalation anesthesia, the calvarial skin was depilated and prepared. After local disinfection, a U-shaped skin incision about 4 cm long was made. After turning over the skin, a vertical periosteal incision about 2 cm long was made along the midline. The periosteum was separated, and circular calvarial bone defects with an inner diameter of 5 mm were made on both sides of the midline, taking care not to damage the dura mater.

[0053] Example 4

[0054] Verification of the effectiveness of bone marrow mesenchymal stem cell membrane sheets using an animal model

[0055] The stacked three-layer bone marrow mesenchymal stem cell membrane sheets were placed into the bone defect on the left calvaria of the rats. The right side served as the control group. The periosteum and skin were sutured layer by layer. Immediately after the operation, penicillin was intramuscularly injected to prevent postoperative infection. Micro-CT scans were performed at 4 weeks and 8 weeks after the operation to observe the repair of the local bone defect, and local samples were taken for fixation and histological detection of osteogenesis.

Claims

1. A human mesenchymal stem cell membrane sheet, characterized in that, Comprising the following steps: Preparation of stem cell membrane sheets The seeding density of human mesenchymal stem cells cultured in vitro can be 1×105 - 2×105 cells / cm2. They are seeded into a conventional culture dish or culture plate and cultured in a basal medium conventionally for 24 h, and then changed to a special medium, with the medium changed once every 3 days.

2. According to claim 1, the basal medium comprises: α-MEM, 10 v / v% fetal bovine serum (FBS), 1 v / v% penicillin-streptomycin (PS). The special medium includes: basal medium, 1 v / v% ITS, 0.04 μg / mL dexamethasone, 15 mM HEPES, 50 μg / mL vitamin C.

3. As described in claim 1, after the stem cells are cultured in the special medium for 1 - 7 days, the edge of the culture dish is gently and repeatedly pipetted to detach the cells at the edge from the bottom of the culture dish, and they gradually detach as a whole to form a complete cell membrane sheet.

4. For a monolayer stem cell membrane sheet prepared by the stem cell preparation method obtained by any one of the preparation methods described in claims 1 - 3, remove the medium and lay it flat on the bottom of the culture dish, and let it stand for 20 minutes at 37 °C and 5% CO2 (the first cell membrane sheet). Use a pipette to slowly place the second cell membrane sheet that has been completely peeled off and contains a small amount of medium above the first cell membrane sheet, and let it fully spread and cover the first cell membrane sheet. Then remove the medium again and let it stand for 20 minutes at 37 °C and 5% CO2. Place the third cell membrane sheet above the second cell membrane sheet in the same way, and remove the medium again and let it stand for 20 minutes at 37 °C and 5% CO2.

Images