Amniotic mesenchymal stem cell injection and large-scale construction method thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an amniotic mesenchymal stem cell injection and a large-scale construction method thereof. The amniotic mesenchymal stem cell injection is prepared from amniotic mesenchymal stem cells, a compound electrolyte injection, hydroxyethyl starch, low-molecular-weight heparin calcium, vitamin C, trehalose, ophiopogonic acid, ganoderma capense glycopeptide and water. The amniotic mesenchymal stem cell injection provided by the invention can effectively maintain the cell viability and osteogenic differentiation capacity of amniotic mesenchymal stem cells, and additives are clinically common drugs, so that the amniotic mesenchymal stem cell injection is safe and has no side effects.

Description

Technical Field

[0001] The invention belongs to the technical field of cell cryopreservation, and particularly relates to an amniotic mesenchymal stem cell injection and a large-scale construction method thereof. Background Art

[0002] Bone damage or defect caused by trauma, infection, tumor resection, etc. is a common and frequently occurring disease in orthopedics. The current traditional treatments for bone defect repair and functional reconstruction have significant limitations and are difficult to achieve ideal therapeutic effects. In recent years, with the breakthrough progress of stem cell engineering technology, regenerative medicine and tissue engineering with stem cell technology as the core have shown great potential, providing a new solution for bone defect treatment.

[0003] Amniotic mesenchymal stem cells are derived from placental amniotic tissue, have multidirectional differentiation potential, low immunogenicity, and anti-inflammatory properties, and show significant potential in regenerative medicine, immune regulation, and tissue repair. A large number of studies have shown that amniotic mesenchymal stem cells have stronger expansion ability, differentiation ability, and low immunogenicity than bone marrow mesenchymal stem cells, and are the best source of seed cells.

[0004] At present, it has been confirmed that amniotic mesenchymal stem cells can differentiate into osteoblasts under certain conditions. Osteoblasts are the main functional cells of bone formation, responsible for the synthesis, secretion and mineralization of bone matrix. Bone tissue defects caused by their formation and dysfunction are related to orthopedic diseases, such as osteoarthritis and osteoporosis. Therefore, amniotic mesenchymal stem cells have great application prospects in the field of bone tissue defect repair as osteoblasts. The key lies in finding an inducer that is efficient, non-toxic, has no side effects, and has good biocompatibility to promote the osteogenic differentiation of amniotic mesenchymal stem cells.

[0005] Based on the above purpose, the present invention provides an amniotic membrane mesenchymal stem cell injection and a large-scale construction method thereof. Summary of the invention

[0006] The first object of the present invention is to provide an amniotic membrane mesenchymal stem cell injection.

[0007] The second object of the present invention is to provide a method for large-scale construction of amniotic mesenchymal stem cell injection.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is: An amniotic membrane mesenchymal stem cell injection comprises amniotic membrane mesenchymal stem cells, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, ophiopogon acid, bozhi glycopeptide, and water.

[0009] Further, the final cell concentration of amniotic mesenchymal stem cells in the amniotic mesenchymal stem cell injection is 1 - 3×10 7 cells / mL.

[0010] Further, the final cell concentration of amniotic mesenchymal stem cells in the amniotic mesenchymal stem cell injection is 2×10 7 cells / mL.

[0011] Further, the amniotic mesenchymal stem cells are P2 - P5 generation amniotic mesenchymal stem cells.

[0012] Further, in the amniotic mesenchymal stem cell injection, the mass fraction of compound electrolyte injection is 35 - 42%, the mass fraction of hydroxyethyl starch is 10 - 15%, the mass fraction of low molecular weight heparin calcium is 3 - 8%, the mass fraction of vitamin C is 1 - 3%, the mass fraction of trehalose is 0.5 - 1.5%, the mass fraction of triplaris acid is 0.2 - 0.4%, and the mass fraction component of ganoderma lucidum polysaccharide peptide is 0.3 - 0.5%.

[0013] Further, in the amniotic mesenchymal stem cell injection, the mass fraction of compound electrolyte injection is 38%, the mass fraction of hydroxyethyl starch is 12%, the mass fraction of low molecular weight heparin calcium is 5%, the mass fraction of vitamin C is 2%, the mass fraction of trehalose is 1.0%, the mass fraction of triplaris acid is 0.3%, and the mass fraction component of ganoderma lucidum polysaccharide peptide is 0.4%.

[0014] The large-scale construction method of the above-mentioned amniotic mesenchymal stem cell injection is as follows: (1) Take compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, triplaris acid, and ganoderma lucidum polysaccharide peptide, add them to water according to the corresponding usage amounts of each raw material component, fully dissolve and mix them, and then filter and sterilize with a filter to obtain a mixed solution for standby; (2) Resuspend the amniotic mesenchymal stem cells in the mixed solution of step (1) to obtain the amniotic mesenchymal stem cell injection.

[0015] Further, the pore size of the filter is 0.22 μm.

[0016] Compared with the prior art, the beneficial effects of the present invention mainly lie in: The present invention prepares an amniotic mesenchymal stem cell injection solution, and the amniotic mesenchymal stem cell injection solution includes amniotic mesenchymal stem cells, compound electrolyte injection solution, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, scirpusin A, ganoderma lucidum polypeptide, and water. In the amniotic mesenchymal stem cell injection solution provided by the present invention, scirpusin A and ganoderma lucidum are added, which avoids the side effects brought by human albumin in traditional injection solutions. At the same time, scirpusin A and ganoderma lucidum polypeptide have a synergistic effect, which can effectively maintain the viability of amniotic mesenchymal stem cells while also maintaining the osteogenic differentiation ability of amniotic mesenchymal stem cells, and the additives are all commonly used drugs in clinical practice, which are safe and have no side effects. Description of the Drawings

[0017] Figure 1 It is a morphological diagram of passage 3 amniotic mesenchymal stem cells; Figure 2 It is a morphological diagram of passage 2 amniotic mesenchymal stem cells; Figure 3 It is a morphological diagram of passage 4 amniotic mesenchymal stem cells; Figure 4 It is a result diagram of the influence of the amniotic mesenchymal stem cell injection solution prepared in Examples 1-3 of the present invention on the osteogenic differentiation of amniotic mesenchymal stem cells; In the figure, a represents Example 1; b represents Example 2; c represents Example 3; Figure 5 It is a result diagram of the influence of the amniotic mesenchymal stem cell injection solution prepared in Comparative Examples 1-4 of the present invention on the osteogenic differentiation of amniotic mesenchymal stem cells; In the figure, a represents Comparative Example 1; b represents Comparative Example 2; c represents Comparative Example 3; d represents Comparative Example 4. Detailed Embodiments

[0018] The following content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can be made, and all should be regarded as belonging to the protection scope of the present invention. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are all conventional products obtained through commercial channels.

[0019] Example 1 An amniotic mesenchymal stem cell injection solution, the raw material components include: amniotic mesenchymal stem cells, compound electrolyte injection solution, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, scirpusin A, ganoderma lucidum polypeptide, and water.

[0020] Among them, the mass fraction of compound electrolyte injection in the amniotic mesenchymal stem cell injection is 38%, the mass fraction of hydroxyethyl starch is 12%, the mass fraction of low molecular weight heparin calcium is 5%, the mass fraction of vitamin C is 2%, the mass fraction of trehalose is 1.0%, the mass fraction of trichosanthemi acid is 0.3%, the mass fraction component of ganoderma lucidum polypeptide is 0.4%, and the final cell concentration of amniotic mesenchymal stem cells is 2×10 7 cells / mL.

[0021] A method for large-scale construction of an amniotic mesenchymal stem cell injection, and the specific large-scale construction method is as follows: (1) Isolation and culture of human amniotic mesenchymal stem cells: Mechanically peel the amniotic membrane from a fresh placenta of a healthy full-term cesarean section, rinse and cut into pieces; Digest twice with trypsin digestion solution containing 0.02% EDTA, remove the digestion solution of amniotic epithelial cells, and retain the mesenchymal stem cell-enriched tissue. The remaining tissue is digested with type II collagenase + DNase I for 2 h, and the amniotic tissue fragments are completely dissociated into a flocculent cell suspension; After filtration and centrifugation, a cell pellet is obtained, and the obtained pellet is the P0 generation of human amniotic mesenchymal stem cells. Resuspend the amniotic mesenchymal stem cells with low-glucose DMEM containing 10% FBS, inoculate them into a T25 flask, and perform primary cell culture in a constant temperature incubator at 37 °C and 5% CO2. After 48 h of culture, observe the cell attachment under a microscope and replace the fresh medium for the cells. Wait until the cell culture reaches 80% confluence and then perform passage culture. The passage ratio is 1:2 to obtain the P3 generation of amniotic mesenchymal stem cells, and use a microscope to observe the morphology of the amniotic mesenchymal stem cells. The results are as Figure 1 shown.

[0022] (2) According to the usage amounts of the respective raw material components, add compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, trichosanthemi acid, and ganoderma lucidum polypeptide to water, fully dissolve and mix them, and then filter and sterilize them with a filter with a pore size of 0.22 μm to obtain a mixed solution for standby.

[0023] (3) Add the P3 generation of amniotic mesenchymal stem cells prepared in step (1) to the mixed solution in step (2) and resuspend them to obtain the amniotic mesenchymal stem cell injection. Store and transport the stem cell injection in a dark environment at 0-8 °C.

[0024] Example 2 An amniotic mesenchymal stem cell injection, the raw material components include: amniotic mesenchymal stem cells, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, trichosanthemi acid, ganoderma lucidum polypeptide, and water.

[0025] Among them, the mass fraction of compound electrolyte injection in the amniotic mesenchymal stem cell injection is 35%, the mass fraction of hydroxyethyl starch is 10%, the mass fraction of low molecular weight heparin calcium is 3%, the mass fraction of vitamin C is 1%, the mass fraction of trehalose is 0.5%, the mass fraction of trichosanthin acid is 0.24%, the mass fraction component of ganoderma lucidum polysaccharide peptide is 0.3%, and the final cell concentration of amniotic mesenchymal stem cells is 1×10 7 cells / mL.

[0026] A method for large-scale construction of an amniotic mesenchymal stem cell injection, and the specific large-scale construction method is as follows: (1) The culture method of amniotic mesenchymal stem cells is the same as that in Example 1 to obtain P2-generation human amniotic mesenchymal stem cells. Use a microscope to observe the morphology of amniotic mesenchymal stem cells, and the results are as Figure 2 shown.

[0027] (2) According to the usage amounts of each raw material component, add compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, trichosanthin acid, and ganoderma lucidum polysaccharide peptide into water, fully dissolve and mix them, and then filter and sterilize with a filter with a pore size of 0.22 μm to obtain a mixed solution for standby.

[0028] (3) Add the P2-generation amniotic mesenchymal stem cells prepared in step (1) into the mixed solution in step (2) for resuspension to obtain an amniotic mesenchymal stem cell injection. Store and transport the stem cell injection in a dark environment at 0-8°C.

[0029] Example 3 An amniotic mesenchymal stem cell injection, the raw material components include: amniotic mesenchymal stem cells, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, trichosanthin acid, ganoderma lucidum polysaccharide peptide, and water.

[0030] Among them, the mass fraction of compound electrolyte injection in the amniotic mesenchymal stem cell injection is 42%, the mass fraction of hydroxyethyl starch is 15%, the mass fraction of low molecular weight heparin calcium is 8%, the mass fraction of vitamin C is 3%, the mass fraction of trehalose is 1.5%, the mass fraction of trichosanthin acid is 0.4%, the mass fraction component of ganoderma lucidum polysaccharide peptide is 0.5%, and the final cell concentration of amniotic mesenchymal stem cells is 3×10 7 cells / mL.

[0031] A method for large-scale construction of an amniotic mesenchymal stem cell injection, and the specific large-scale construction method is as follows: (1) The culture method of amniotic mesenchymal stem cells is the same as that in Example 1 to obtain P4-generation human amniotic mesenchymal stem cells. Use a microscope to observe the morphology of amniotic mesenchymal stem cells, and the results are as Figure 3 shown.

[0032] (2) According to the usage amounts corresponding to each raw material component, add compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, scillarenic acid, and ganoderma lucidum polypeptide to water, fully dissolve and mix them, and then filter and sterilize with a filter with a pore size of 0.22 μm to obtain a mixed solution for standby.

[0033] (3) Resuspend the P4 generation amniotic mesenchymal stem cells prepared in step (1) in the mixed solution of step (2) to obtain an amniotic mesenchymal stem cell injection, and store and transport the stem cell injection in a light-proof environment at 0 - 8°C.

[0034] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that scillarenic acid is omitted from the components of the mixed solution prepared in step (2), and the others are the same as in Example 1.

[0035] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that ganoderma lucidum polypeptide is omitted from the components of the mixed solution prepared in step (2), and the others are the same as in Example 1.

[0036] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that scillarenic acid is omitted from the components of the mixed solution prepared in step (2), and the dosage of ganoderma lucidum polypeptide is adjusted to the sum of the two.

[0037] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that ganoderma lucidum polypeptide is omitted from the components of the mixed solution prepared in step (2), and the dosage of scillarenic acid is adjusted to the sum of the two.

[0038] Test Example 1 Comparison of cell viability in different amniotic mesenchymal stem cell injections Place the amniotic mesenchymal stem cell injections prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 of the present invention in a temperature environment of 4°C for storage. At 4, 8, 12, 24, 36, and 48 hours of storage, respectively take 0.5 mL of the amniotic mesenchymal stem cell injection and measure the cell survival rate. The specific measurement steps are as follows: (1) Take 0.5 mL of the amniotic mesenchymal stem cell injection and add it to a test tube, and add 0.5 mL of a trypan blue dye solution with a concentration of 0.2% to the test tube to obtain a mixed solution; (2) Use a pipette to aspirate 10 μL of the mixed solution in step (1) and drop it on the edge of a coverslip to ensure that the suspension fills the space between the coverslip and the counting plate; (3) Leave the prepared sample to stand for 1 minute to allow the cells to fully interact with the staining solution. Randomly select several fields of view and count a total of 300 cells, and determine the number of dead cells among them. According to the counting results, calculate the cell viability. The cell viability (%) = [(number of live cells) / (number of live cells + number of dead cells)] × 100%. The results are shown in Table 1.

[0039] Table 1 Cell viability The results are shown in Table 1, which are the cell viabilities of the amniotic mesenchymal stem cell injections in Examples 1-3 and Comparative Examples 1-4. Compared with the groups of Comparative Examples 1-4, the amniotic mesenchymal stem cell injections prepared in Examples 1-3 of the present invention have a higher preservation rate of stem cells and a higher cell viability. It shows that the synergistic effect of juncusol and ganoderma lucidum polysaccharide peptide can effectively maintain the activity of human amniotic mesenchymal stem cells.

[0040] Test Example 2 Effect of amniotic mesenchymal stem cell injection on osteogenic differentiation of amniotic mesenchymal stem cells (1) After storing the amniotic mesenchymal stem cell injections prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 of the present invention at 4 °C for 4 hours, centrifuge and discard the supernatant to obtain amniotic mesenchymal stem cells. Add 500 μL of a gelatin coating solution with a concentration of 0.1% to the bottom of a 24-well plate, incubate at 37 °C for 1 h, then aspirate the gelatin after completion and air-dry for later use.

[0041] (2) Seed the amniotic mesenchymal stem cells at 2×10 4 / well into the 24-well culture plate obtained in step (1), and culture the cells in DMEM / F12 medium containing 10% (v / v) FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL) until the cell confluence reaches about 70%. Then discard the supernatant and place it in an osteogenic induction differentiation medium (DMEM / F12 medium containing dexamethasone (0.15 μmol / L), β-glycerophosphate sodium (10 mmol / L), sodium ascorbate (50 mg / L), 10% (v / v) FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL)) to induce the osteogenic differentiation of amniotic mesenchymal stem cells.

[0042] (3)Replace the osteogenic induction medium every 2 days. On the 8th day of osteogenic induction culture, take out the 24-well plate from the incubator and place it in the laminar flow hood. After removing the original medium in the wells, wash 3 times with PBS. Add 500 μL of paraformaldehyde solution to fix for 15 min, then discard the fixative and wash 3 times with PBS. According to the alkaline phosphatase kit instructions, stain the cells of amniotic mesenchymal stem cell osteogenic differentiation obtained. After staining, take pictures under a fluorescence microscope, and judge the osteogenic differentiation ability of amniotic mesenchymal stem cells according to the staining results.

[0043] The results are as Figure 4 , Figure 5 shown, which are the result diagrams of the effects of different amniotic mesenchymal stem cell injections on the osteogenic differentiation of human amniotic mesenchymal stem cells. Compared with Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, the amniotic mesenchymal stem cell injections prepared in Examples 1-3 of the present invention can maintain the osteogenic differentiation ability of amniotic mesenchymal stem cells. It shows that the synergistic effect of trichophyllic acid and ganodermapolysaccharide peptide can effectively maintain the activity of amniotic mesenchymal stem cells, thereby maintaining the osteogenic differentiation ability of amniotic mesenchymal stem cells.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described in the above with specific implementation schemes. On the basis of the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of protection required by the present invention.

Claims

1. An amniotic mesenchymal stem cell injection, characterized in that, The amniotic mesenchymal stem cell injection includes amniotic mesenchymal stem cells, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, trichosanthin, ganoderma lucidum polypeptide, and water.

2. The amniotic mesenchymal stem cell injection according to claim 1, characterized in that, The final cell concentration of amniotic mesenchymal stem cells in the amniotic mesenchymal stem cell injection is 1 - 3×10 7 cells / mL.

3. The amniotic mesenchymal stem cell injection according to claim 2, wherein The final cell concentration of amniotic mesenchymal stem cells in the amniotic mesenchymal stem cell injection is 2×10 7 cells / mL.

4. The amniotic mesenchymal stem cell injection according to claim 3, characterized in that, The amniotic mesenchymal stem cells are amniotic mesenchymal stem cells at passages P2 - P5.

5. The amniotic mesenchymal stem cell injection according to claim 1, wherein In the amniotic mesenchymal stem cell injection, the mass fraction of the compound electrolyte injection is 35 - 42%, the mass fraction of the hydroxyethyl starch is 10 - 15%, the mass fraction of the low molecular weight heparin calcium is 3 - 8%, the mass fraction of the vitamin C is 1 - 3%, the mass fraction of the trehalose is 0.5 - 1.5%, the mass fraction of the trichosanthin is 0.2 - 0.4%, and the mass fraction component of the ganoderma lucidum polypeptide is 0.3 - 0.5%.

6. The amniotic mesenchymal stem cell injection according to claim 5, wherein In the amniotic mesenchymal stem cell injection, the mass fraction of the compound electrolyte injection is 38%, the mass fraction of the hydroxyethyl starch is 12%, the mass fraction of the low molecular weight heparin calcium is 5%, the mass fraction of the vitamin C is 2%, the mass fraction of the trehalose is 1.0%, the mass fraction of the trichosanthin is 0.3%, and the mass fraction component of the ganoderma lucidum polypeptide is 0.4%.

7. The large-scale construction method of the amniotic mesenchymal stem cell injection according to any one of claims 1-6, characterized in that, The specific large - scale construction method is as follows: (1) Take the compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, trichosanthin, ganoderma lucidum polypeptide, and add them to water according to the corresponding usage amounts of each raw material component, fully dissolve and mix them, and then filter and sterilize with a filter to obtain a mixed solution for standby; (2) Resuspend the amniotic mesenchymal stem cells in the mixed solution of step (1) to obtain the amniotic mesenchymal stem cell injection.

8. The large-scale construction method of the amniotic mesenchymal stem cell injection according to claim 7, characterized in that, The pore size of the filter is 0.22 μm.

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