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

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an amniotic membrane mesenchymal stem cell injection and a large-scale construction method thereof. The 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, acid of water ophiopogon japonicus, and thin zhisugar peptide, and water. The amniotic membrane mesenchymal stem cell injection can effectively maintain the cell survival rate and osteogenic differentiation capacity of the amniotic membrane mesenchymal stem cells, and the additives are all commonly used drugs in the clinic, and are safe and have no side effects.

Description

TECHNICAL FIELD

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

[0002] Bone damage or defect caused by trauma, infection, tumor resection and other reasons is a common and frequently-occurring disease in orthopedic diseases. The current traditional treatment for bone defect repair and functional reconstruction has significant limitations and is difficult to achieve ideal treatment effects. In recent years, with the breakthrough progress of stem cell engineering technology, regenerative medicine and tissue engineering based on stem cell technology have shown great potential and provided a new solution for bone defect treatment.

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

[0004] At present, amniotic membrane mesenchymal stem cells have been proved to be able to 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, and the formation and dysfunction of osteoblasts cause bone tissue defects and orthopedic diseases such as osteoarthritis and osteoporosis. Therefore, amniotic membrane mesenchymal stem cells as osteoblasts have great application prospects in the field of bone tissue defect repair. The key is to find an efficient, non-toxic, non-side-effect and biocompatible inducer for promoting the osteogenic differentiation of amniotic membrane mesenchymal stem cells.

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

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

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

[0008] In order to achieve the above object, the technical scheme adopted by the application is:

[0009] An amniotic membrane mesenchymal stem cell injection, which comprises amniotic membrane mesenchymal stem cells, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, water ophiopogon japonicus acid, tianzhi glycopeptide and water.

[0010] Further, the cell final concentration of the amniotic membrane mesenchymal stem cell injection is 1-3×10 7 .

[0011] Further, the cell final concentration of the amniotic membrane mesenchymal stem cell injection is 2×10 7 .

[0012] Further, the amniotic membrane mesenchymal stem cell is P2-P5 generation amniotic membrane mesenchymal stem cell.

[0013] Further, the mass fraction of the compound electrolyte injection in the amniotic membrane mesenchymal stem cell 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 water ophiopogon japonicus acid is 0.2-0.4%, and the mass fraction of the thin zhi glycopeptide is 0.3-0.5%.

[0014] Further, the mass fraction of the compound electrolyte injection in the amniotic membrane mesenchymal stem cell 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 water ophiopogon japonicus acid is 0.3%, and the mass fraction of the thin zhi glycopeptide is 0.4%.

[0015] The above-mentioned amniotic membrane mesenchymal stem cell injection is constructed in a large scale, and the specific large-scale construction method is as follows:

[0016] (1) The compound electrolyte injection, the hydroxyethyl starch, the low molecular weight heparin calcium, the vitamin C, the trehalose, the water ophiopogon japonicus acid and the thin zhi glycopeptide are taken, and are added into water according to the corresponding use amount of each raw material component, and are dissolved and mixed uniformly, and then are filtered by a filter to remove bacteria, so as to obtain a mixed solution for standby;

[0017] (2) The amniotic membrane mesenchymal stem cell is added into the mixed solution of step (1) to resuspend, so as to obtain the amniotic membrane mesenchymal stem cell injection.

[0018] Further, the pore size of the filter is 0.22 μm.

[0019] Compared with the prior art, the beneficial effects of the present application mainly lie in:

[0020] The amniotic membrane mesenchymal stem cell injection prepared by the application comprises amniotic membrane mesenchymal stem cells, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, shuimaidong acid, thin zhisugar peptide and water. The amniotic membrane mesenchymal stem cell injection provided by the application adds shuimaidong acid and thin zhisugar peptide, thereby avoiding the side effects caused by human blood albumin in the traditional injection, and the shuimaidong acid and thin zhisugar peptide have a synergistic effect, which can effectively maintain the viability of the amniotic membrane mesenchymal stem cells and maintain the osteogenic differentiation ability of the amniotic membrane mesenchymal stem cells, and the additives are all commonly used drugs in the clinic, which are safe and have no side effects. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a P3 generation amniotic membrane mesenchymal stem cell morphology diagram;

[0022] Figure 2 It is a P2 generation amniotic membrane mesenchymal stem cell morphology diagram;

[0023] Figure 3 It is a P4 generation amniotic membrane mesenchymal stem cell morphology diagram;

[0024] Figure 4 It is a result diagram of the influence of the amniotic membrane mesenchymal stem cell injection prepared by the examples 1-3 on the osteogenic differentiation of amniotic membrane mesenchymal stem cells;

[0025] In the diagram, a represents example 1, b represents example 2, and c represents example 3.

[0026] Figure 5 It is a result diagram of the influence of the amniotic membrane mesenchymal stem cell injection prepared by the comparative examples 1-4 on the osteogenic differentiation of amniotic membrane mesenchymal stem cells;

[0027] In the diagram, a represents comparative example 1, b represents comparative example 2, c represents comparative example 3, and d represents comparative example 4. DETAILED DESCRIPTION

[0028] The following is a further detailed description of the application in conjunction with specific preferred embodiments, which cannot be deemed to limit the specific implementation of the application to these descriptions. For ordinary skilled persons in the technical field to which the application belongs, a number of simple deductions or substitutions can be made without departing from the concept of the application, which shall be deemed to fall within the protection scope of the application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used, such as the conventional products obtained through the market channel, are not specifically mentioned.

[0029] Example 1

[0030] An amniotic membrane mesenchymal stem cell injection, raw material components include: amniotic membrane mesenchymal stem cells, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, water ophiopogon japonicus acid, thin zhi glycopeptide, water.

[0031] Among them, the mass fraction of compound electrolyte injection in amniotic membrane 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 water ophiopogon japonicus acid is 0.3%, the mass fraction of thin zhi glycopeptide is 0.4%, and the final concentration of amniotic membrane mesenchymal stem cells is 2x10 7 Individual / mL.

[0032] A kind of amniotic membrane mesenchymal stem cell injection scale construction method, specific scale construction method is as follows:

[0033] (1) Isolation and culture of human amniotic membrane mesenchymal stem cells: fresh placenta of healthy full-term cesarean section is mechanically stripped of amnion, washed and cut into small pieces; digest twice with trypsin digestion solution containing 0.02% EDTA, remove the digestion solution of amnion epithelial cells, and retain the mesenchymal stem cell-rich tissue. The remaining tissue is digested with type II collagenase + DNase I for 2 h, and the amnion tissue fragments are completely dissociated into a flocculent cell suspension; after filtration and centrifugation, the cell pellet is obtained. The obtained pellet is P0 generation human amniotic membrane mesenchymal stem cells. Resuspend the amniotic membrane mesenchymal stem cells in low-glucose DMEM containing 10% FBS, inoculate in a T25 bottle, and culture the primary cells in a constant temperature incubator at 37℃ and 5% CO2. After 48 h of culture, observe the cell adhesion under a microscope, and replace the fresh culture medium. When the cells are cultured to 80% confluence, subculture the cells. The subculture ratio is 1:2, and P3 generation amniotic membrane mesenchymal stem cells are obtained. Observe the morphology of amniotic membrane mesenchymal stem cells using a microscope, and the results are shown in Figure 1 .

[0034] (2) According to the corresponding usage of each raw material component, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, water ophiopogon japonicus acid, and thin zhi glycopeptide are added to water and dissolved and mixed thoroughly, and then filtered and sterilized with a filter with a pore size of 0.22 μm to obtain a mixed solution for standby.

[0035] (3) Add P3 generation amniotic membrane mesenchymal stem cells prepared in step (1) to the mixed solution in step (2) to resuspend, and obtain amniotic membrane mesenchymal stem cell injection. Store and transport the stem cell injection in a 0-8℃ dark environment.

[0036] Example 2

[0037] An amniotic membrane mesenchymal stem cell injection, raw material components comprising: amniotic membrane mesenchymal stem cells, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, water ophiopogon japonicus acid, and thin zhi glycopeptide.

[0038] Among them, the mass fraction of compound electrolyte injection in amniotic membrane 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 water ophiopogon japonicus acid is 0.24%, the mass fraction of thin zhi glycopeptide is 0.3%, and the final concentration of amniotic membrane mesenchymal stem cells is 1×10 7 Individuals / mL.

[0039] A method for large-scale construction of an amniotic membrane mesenchymal stem cell injection, and the specific large-scale construction method is as follows:

[0040] (1) The culture method of amniotic membrane mesenchymal stem cells is the same as in Example 1, and P2 generation human amniotic membrane mesenchymal stem cells are obtained. The morphology of amniotic membrane mesenchymal stem cells is observed using a microscope, and the results are shown in Figure 2 .

[0041] (2) According to the corresponding usage of each raw material component, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, water ophiopogon japonicus acid, and thin zhi glycopeptide are added to water and fully dissolved and mixed, and then filtered and sterilized with a filter with a pore size of 0.22 μm to obtain a mixed solution for standby.

[0042] (3) The P2 generation amniotic membrane mesenchymal stem cells prepared in step (1) are resuspended in the mixed solution of step (2) to obtain an amniotic membrane mesenchymal stem cell injection. The stem cell injection is stored and transported in a light-proof environment at 0-8℃.

[0043] Example 3

[0044] An amniotic membrane mesenchymal stem cell injection, raw material components comprising: amniotic membrane mesenchymal stem cells, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, water ophiopogon japonicus acid, and thin zhi glycopeptide.

[0045] Among them, the mass fraction of compound electrolyte injection in amniotic membrane 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 water ophiopogon japonicus acid is 0.4%, the mass fraction of thin zhi glycopeptide is 0.5%, and the final concentration of amniotic membrane mesenchymal stem cells is 3×10 7 Individuals / mL.

[0046] A large-scale construction method of amniotic membrane mesenchymal stem cell injection, the specific large-scale construction method is as follows:

[0047] (1) The culture method of amniotic membrane mesenchymal stem cells is the same as that in Example 1, and P4 generation of human amniotic membrane mesenchymal stem cells is obtained. The morphology of amniotic membrane mesenchymal stem cells is observed using a microscope, and the results are shown in Figure 3 .

[0048] (2) According to the corresponding use amount of each raw material component, compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, water maidong acid, and thin zhi sugar peptide are added to water and dissolved and mixed uniformly, and then filtered and sterilized with a filter with a pore size of 0.22 μm to obtain a mixed solution for standby.

[0049] (3) The P4 generation of amniotic membrane mesenchymal stem cells prepared in step (1) are resuspended in the mixed solution of step (2) to obtain amniotic membrane mesenchymal stem cell injection. The stem cell injection is stored and transported in a light-proof environment at 0-8℃.

[0050] Comparative Example 1

[0051] The difference between Comparative Example 1 and Example 1 is that the mixed solution prepared in step (2) is missing water maidong acid, and the others are the same as Example 1.

[0052] Comparative Example 2

[0053] The difference between Comparative Example 2 and Example 1 is that the mixed solution prepared in step (2) is missing thin zhi sugar peptide, and the others are the same as Example 1.

[0054] Comparative Example 3

[0055] The difference between Comparative Example 3 and Example 1 is that the mixed solution prepared in step (2) is missing water maidong acid, and the amount of thin zhi sugar peptide is adjusted to the sum of the two.

[0056] Comparative Example 4

[0057] The difference between Comparative Example 4 and Example 1 is that the mixed solution prepared in step (2) is missing thin zhi sugar peptide, and the amount of water maidong acid is adjusted to the sum of the two.

[0058] Test Example 1

[0059] Comparison of cell viability in different amniotic membrane mesenchymal stem cell injections

[0060] The amniotic membrane mesenchymal stem cell injection solutions prepared in Examples 1, 2, 3, 1, 2, 3, and 4 of the present invention were stored at 4°C. After 4, 8, 12, 24, 36, and 48 hours, 0.5 mL of the amniotic membrane mesenchymal stem cell injection solution was taken and the cell viability was measured. The specific measurement steps are as follows:

[0061] (1) Take 0.5 mL of amniotic membrane mesenchymal stem cell injection solution and add it to a test tube. Then add 0.5 mL of 0.2% trypan blue dye solution to the test tube to obtain a mixed solution.

[0062] (2) Use a pipette to aspirate 10 μL of the mixed solution from step (1) and drop it onto the edge of the coverslip, ensuring that the suspension fills the space between the coverslip and the counting plate;

[0063] (3) Let the prepared sample stand for 1 minute to allow the cells to fully react with the dye. Randomly select several fields of view and count a total of 300 cells to determine the number of dead cells. Based on the counting results, calculate the cell viability (%) = [(number of live cells) / (number of live cells + number of dead cells)] × 100%. The results are shown in Table 1.

[0064] Table 1 Cell viability

[0065]

[0066] The results are shown in Table 1, which shows the cell viability of the amniotic membrane mesenchymal stem cell injections prepared in Examples 1-3 and Comparative Examples 1-4. Compared with Comparative Examples 1-4, the amniotic membrane mesenchymal stem cell injections prepared in Examples 1-3 of the present invention exhibited a higher stem cell preservation rate and higher cell viability. This indicates that the synergistic effect of ophiopogon acid and glycosaminoglycans can effectively maintain the activity of human amniotic membrane mesenchymal stem cells.

[0067] Test Example 2

[0068] Effect of amniotic mesenchymal stem cell injection on osteogenic differentiation of amniotic mesenchymal stem cells

[0069] (1) The amniotic membrane mesenchymal stem cell injection solutions prepared in Examples 1, 2, 3, 1, 2, 3, and 4 of the present invention were stored at 4°C for 4 hours, centrifuged, and the supernatant discarded to obtain amniotic membrane mesenchymal stem cells. 500 μL of 0.1% gelatin coating solution was added to the bottom of a 24-well plate and incubated at 37°C for 1 hour. After incubation, the gelatin was aspirated and dried for later use.

[0070] (2) Amniotic mesenchymal stem cells were cultured at a rate of 2×10 4 / well were seeded into the 24-well culture plate obtained in step (1), and the cells were cultured in DMEM / F12 medium containing 10% (v / v) FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL) until the cells were about 70% confluent, the supernatant was discarded, and the cells were placed in osteogenic differentiation induction medium (DMEM / F12 medium containing dexamethasone (0.15 μmol / L), sodium β-glycerophosphate (10 mmol / L), sodium ascorbate (50 mg / L), 10% (v / v) FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL)) to induce osteogenic differentiation of amniotic mesenchymal stem cells.

[0071] (3) Replace the osteogenic induction medium every 2 days. On the 8th day of osteogenic induction culture, remove the 24-well plate from the incubator and place it on the clean bench. Remove the original culture medium in the wells and wash with PBS 3 times. Add 500 μL of paraformaldehyde solution and fix for 15 minutes. Discard the fixative and wash with PBS 3 times. According to the instructions of the alkaline phosphatase kit, the osteogenic differentiated cells of the obtained amniotic mesenchymal stem cells were stained. After staining, take photos under a fluorescence microscope. The osteogenic differentiation ability of the amniotic mesenchymal stem cells was determined based on the staining results.

[0072] The results are as follows Figure 4 、 Figure 5 The figure shows the effects of different amniotic mesenchymal stem cell injections on the osteogenic differentiation of human amniotic mesenchymal stem cells. Compared with Comparative Examples 1, 2, 3, and 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. This shows that the synergistic effect of ophiopogon acid and glycosaminoglycans can effectively maintain the activity of amniotic mesenchymal stem cells, thereby maintaining the osteogenic differentiation ability of amniotic mesenchymal stem cells.

[0073] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. An amniotic membrane mesenchymal stem cell injection, characterized in that: The 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, hydrolyzed ophiopogon acid, glycoside peptide, and water; the final concentration of the amniotic membrane mesenchymal stem cells in the amniotic membrane mesenchymal stem cell injection is 1-3×10 7 / mL; the mass fraction of the compound electrolyte injection in the amniotic membrane mesenchymal stem cell 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 liuopogon acid is 0.2-0.4%, and the mass fraction of the glycosaminoglycan component is 0.3-0.5%.

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

3. The amniotic membrane mesenchymal stem cell injection according to claim 2, characterized in that: The amniotic mesenchymal stem cells are P2-P5 generation amniotic mesenchymal stem cells.

4. The amniotic membrane mesenchymal stem cell injection according to claim 1, characterized in that: The mass fraction of the compound electrolyte injection in the amniotic membrane 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 ophiopogon acid is 0.3%, and the mass fraction of the bozhi glycopeptide component is 0.4%.

5. The method for large-scale construction of amniotic mesenchymal stem cell injection according to any one of claims 1 to 4, characterized in that: The specific scale construction method is as follows: (1) Take compound electrolyte injection, hydroxyethyl starch, low molecular weight heparin calcium, vitamin C, trehalose, hydrolyzed ophiopogon acid, and glycosaminoglycans according to the corresponding usage amount of each raw material component, add them into water and fully dissolve and mix them, then filter and sterilize them with a filter to obtain a mixed solution for use; (2) Add the amniotic membrane mesenchymal stem cells to the mixed solution of step (1) and resuspend them to obtain an amniotic membrane mesenchymal stem cell injection solution.

6. The method for large-scale construction of amniotic mesenchymal stem cell injection according to claim 5, characterized in that: The pore size of the filter is 0.22 μm.

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