Amniotic membrane preserving fluid and application thereof

Abstract

The invention provides amniotic membrane preserving fluid and application thereof. The amniotic membrane preserving fluid is prepared from the following components: sodium chloride injection, glycerol, trehalose and sodium hyaluronate, the volume ratio of the sodium chloride injection to the glycerol is (8-24): (8-18); the volume mass ratio of the sodium chloride injection to the trehalose is (8-24): (0.5-6); the mass ratio of the trehalose to the sodium hyaluronate is (0.5-6): (0.006-0.15). Compared with an existing amniotic membrane preservation method, the amniotic membrane preservation liquid can be stored at a high temperature, so that the risk that the amniotic membrane is frostbitten can be effectively reduced, even if the amniotic membrane is stored at the high temperature, the amniotic membrane can have high biological activity and multidirectional differentiation potential, and various natural active ingredients of the amniotic membrane can be effectively reserved. Meanwhile, the amniotic membrane preserving fluid is simple in component, has good compatibility with amniotic membrane tissues, does not generate adverse effects on the structure and the function of the amniotic membrane, and has higher biological safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue cell preservation, and particularly relates to an amniotic membrane preservation solution and its application. Background Art

[0002] The amniotic membrane is the innermost layer of the placenta, which is similar in tissue structure to the human conjunctiva. It contains substances required for the growth of ocular surface epithelial cells, including conjunctival cells and corneal epithelial cells. It is smooth, without blood vessels, nerves, and lymphatics, and has a certain elasticity, with a thickness of about 0.02 - 0.5 mm. Under an electron microscope, it is divided into five layers: the epithelial layer, the basement membrane, the dense layer, the fibroblast layer, and the sponge layer. The amniotic basement membrane and the stromal layer contain a large amount of different collagens, mainly type I, III, IV, V, VII collagens, fibronectin, laminin, and other components.

[0003] At present, amniotic membrane transplantation, as a new technology for ocular surface reconstruction, has been widely used in clinical practice. The clinical effect is directly related to the integrity of the structure of the selected amniotic membrane itself and the content of its active factors. Fresh amniotic membrane, because it has a complete epithelial layer, can immediately reconstruct the ocular surface epithelial tissue. The amniotic epithelial cells with active cells can also synthesize cytokines, which may play an active role in the process of ocular surface reconstruction. In addition, in the market of wound repair, biological amniotic membrane can be used to solve the repair problems of skin burns, skin ulcers, diabetic foot, etc., accelerate the repair, and inhibit scar formation. In the field of orthopedics, biological amniotic membrane can repair bone defects, cartilage injuries, and tendon injuries.

[0004] However, fresh human amniotic membrane cannot meet the immediate clinical needs. Therefore, it is necessary to explore a method for long-term and effective preservation of the biological activity of amniotic membrane. Among many preservation methods, the method of slow freezing in liquid nitrogen after pretreatment with antifreeze is relatively effective, but it has disadvantages such as multiple processes, easy contamination, and the need for special equipment, making it difficult to promote. And the temperature of the liquid nitrogen freezing method is usually very low. The research on cryopreserving amniotic membrane at deep low temperature has shown that the epithelial cell membrane of the amniotic membrane is damaged, the number of intracellular vacuoles increases, resulting in the separation and autolysis of the epithelium and the basement membrane, thus destroying the integrity of the amniotic membrane structure and affecting its physiological function.

[0005] At present, most of the amniotic membranes used clinically in China are taken from fresh placentas. After the amniotic membrane is peeled off and processed through standard procedures, it is used for amniotic membrane transplantation surgery on patients' eyes. This often delays the treatment of clinical emergency patients due to the inability to find placentas at that time and the time-consuming donor detection, and there is a great waste due to single use. If there is a simple preservation method, it will greatly facilitate the clinical treatment needs. Therefore, developing a preservation method for fresh amniotic membrane is of great significance for the current clinical needs. Summary of the Invention

[0006] To solve the problems and deficiencies existing in the prior art, the present invention provides an amniotic membrane preservation solution and its application. Compared with the existing amniotic membrane preservation methods, this amniotic membrane preservation solution can be stored at a relatively high temperature, thus effectively reducing the risk of the amniotic membrane being frostbitten. Even when stored at a relatively high temperature, the amniotic membrane can still have high biological activity and multi-directional differentiation potential, that is, it can effectively retain various natural active components of the amniotic membrane. At the same time, the composition of this amniotic membrane preservation solution is simple, has good compatibility with the amniotic membrane tissue, will not have an adverse impact on the structure and function of the amniotic membrane, and has higher biological safety.

[0007] According to the first aspect of the present invention, there is provided an amniotic membrane preservation solution, comprising the following components: sodium chloride injection, glycerol, trehalose, sodium hyaluronate; the volume ratio of sodium chloride injection to glycerol is 8 - 24:8 - 18; the volume-mass ratio of sodium chloride injection to trehalose is 8 - 24:0.5 - 6; the mass ratio of trehalose to sodium hyaluronate is 0.5 - 6:0.006 - 0.15.

[0008] Currently, amniotic membrane preservation solutions have the following characteristics: (1) Complex composition. The main components of amniotic membrane preservation solutions include compound electrolytes, trehalose, dextran, amifostine, mesna, glycerol, polyene phosphatidylcholine, defibrotide, vitamin C, thrombin, recombinant dispase, and recombinant collagenase. These components work together to help preserve the cell viability of amniotic membrane tissue in the long term. However, the complex composition increases the possibility of the presence of immunogenic substances, that is, it contains more potential antigenic components. When the amniotic membrane is used for transplantation or other medical purposes, these antigens may stimulate the body's immune system and trigger an immune response. Moreover, the multiple components mean that more variables need to be monitored and managed during the production process, which makes it difficult to unify the quality standards of the preservation solution, and minor differences in the production process may affect the quality of the final product. This may lead to inconsistencies in the cell viability after membrane preservation, the retention degree of bioactive components, etc., increasing the risk of clinical use. In addition, the complex composition may result in the presence of some potentially toxic substances in the preservation solution, and these toxic substances may damage the amniotic membrane tissue itself or have an adverse impact on human tissues after the amniotic membrane is applied to the human body. (2) Usually, a certain amount of organic solvent such as DMSO needs to be added to lower the freezing point and reduce ice crystal formation. However, organic solvents usually have a certain toxicity, which is not conducive to the safety of use. (3) Usually, liquid nitrogen storage is required. On the one hand, liquid nitrogen storage represents storage at a lower temperature, which will increase the risk of frostbite of the amniotic membrane, and is not conducive to maintaining the original biological activity and structural stability of the amniotic membrane. On the other hand, liquid nitrogen storage requires the equipped liquid nitrogen tanks and liquid nitrogen supply, which are relatively difficult to popularize and inconvenient for transportation. In addition, during the transportation of liquid nitrogen storage, vibration is likely to cause rupture and it is fragile. (4) Complicated operation. Some preservation solutions require complicated processing steps during use, such as special treatment of the amniotic membrane before preservation, or operations such as thawing and dilution of the preservation solution before use, increasing the complexity and time cost of clinical use.

[0009] In the amniotic membrane preservation solution provided by the present invention, firstly, its composition is very simple, only containing several components such as sodium chloride injection, glycerol, trehalose, and sodium hyaluronate, and there are only three active components, namely glycerol, trehalose, and sodium hyaluronate. That is, it effectively reduces the potential antigenic components, reduces immunogenicity, and at the same time reduces the difficulty of quality control, can ensure that the performance differences of the preservation solution are small, reduces the risk of clinical use, and can reduce potentially toxic substances, improving the safety of use. Moreover, the simple composition also greatly reduces the raw material cost. Another important point is that the simple composition is beneficial to the stability of the preservation solution itself, because in the case of complex components, chemical reactions may occur among them, resulting in a decrease in the stability of the preservation solution. These reactions may change the physical and chemical properties such as the pH value and osmotic pressure of the preservation solution, affecting the preservation effect of the amniotic membrane.

[0010] Second, among the above several components, glycerol, as a cryoprotectant, can lower the freezing point of the preservation solution and prevent the formation of ice crystals during low-temperature storage, which may damage the amniotic membrane tissue. Trehalose can be used as a non-permeable protectant for cryopreservation to protect tissues from cryodamage. Sodium hyaluronate can form a protective microenvironment around cells. It can reduce the influence of external factors (such as mechanical damage, chemical substance stimulation, etc.) on the amniotic membrane and improve the storage effect of the amniotic membrane. In addition, there are a large number of hydrophilic groups such as carboxyl and hydroxyl groups on the molecular chain of sodium hyaluronate, and there are also a large number of hydroxyl groups in the trehalose structural formula, and there are also 3 hydroxyl groups in the glycerol structural formula. Therefore, these three substances can not only form hydrogen bonds with water molecules but also form hydrogen bonds with each other, making the entire system of the preservation solution have better water retention performance and further lowering the freezing point of the preservation solution. Therefore, it can further reduce the generation of ice crystals during low-temperature storage and further reduce the cryodamage to the amniotic membrane. Therefore, under the combined action of these three substances, the effective preservation of the amniotic membrane can be achieved at a relatively higher temperature (compared with traditional liquid nitrogen ultra-low temperature preservation), and the amniotic membrane can still maintain a good tissue structure in this preservation solution, and various natural active ingredients are effectively preserved, enabling the amniotic membrane to maintain its original biological structure to the greatest extent, which is beneficial for subsequent clinical use and improves the treatment effect, for example, in the treatment of ocular surface diseases or wound repair. Moreover, the combination of these substances can effectively reduce the adverse chemical reactions between different components and improve the stability of the preservation solution, thus further optimizing the preservation effect of the amniotic membrane.

[0011] Third, the entire amniotic membrane preservation solution does not contain organic solvents such as DMSO, which greatly reduces toxicity and thus greatly improves the biological safety of the preservation solution for use. Only sodium chloride injection is used as the solvent, which greatly reduces the raw material cost.

[0012] Fourth, the preservation solution provided in the present invention can be applicable to storage at a higher temperature compared with traditional liquid nitrogen ultra-low temperature preservation. It can not only reduce the frostbite of the amniotic membrane caused by liquid nitrogen ultra-low temperature preservation, improve the biological activity and the original structural stability of the amniotic membrane, but also greatly reduce the preservation cost. The amniotic membrane can be stored in a refrigerator, and a general household refrigerator can meet the required storage temperature.

[0013] In addition, controlling the proportion of each raw material of the preservation solution within the above range is beneficial for the above several components to interact and present a better preservation effect. It can not only make the preserved amniotic membrane retain humidity and flexibility similar to that of fresh amniotic membrane, ensure that the amniotic membrane has high biological activity, but also is conducive to the stable state of the preservation solution, realizing the long-term freshness preservation of the amniotic membrane, and enabling it to show better treatment effects in later clinical applications, especially showing obvious treatment effects in ocular surface reconstruction. Therefore, the preservation solution provided by the present invention is an amniotic membrane preservation solution with excellent performance.

[0014] Preferably, the volume ratio of sodium chloride injection to glycerol is 10:10; the volume-mass ratio of sodium chloride injection to trehalose is 10:0.684; the mass ratio of trehalose to sodium hyaluronate is 0.684:0.1. Further, controlling the raw material ratios in the amniotic membrane preservation solution within the above ranges is more conducive to these components exerting a better synergistic effect, enabling the amniotic membrane to still possess higher biological activity and higher contents of various natural active components when it is taken out and used again after being preserved in the preservation solution, and only in this way can it exert a better therapeutic effect during clinical use. For example, the preserved amniotic membrane has more obvious effects in anti-inflammation, anti-angiogenesis, anti-scarring, and promoting healing. Especially when the preserved amniotic membrane is used for ocular surface reconstruction, more obvious injury repair effects can be seen.

[0015] Preferably, the concentration of sodium chloride injection is 0.7 - 1.2%. This concentration is the mass concentration, with the unit of g / mL.

[0016] Preferably, the concentration of sodium chloride injection is 0.9%.

[0017] According to the second aspect of the present invention, there is provided an application of the above amniotic membrane preservation solution in preserving amniotic membrane.

[0018] Preferably, during the process of preserving the amniotic membrane, the temperature is -25 to -14°C. During the process of preserving the amniotic membrane with the amniotic membrane preservation solution provided by the present invention, preservation at a higher temperature relative to the ultra-low temperature of liquid nitrogen can be achieved. Therefore, the problem of frostbite of the amniotic membrane can be effectively prevented, and the problem of high toxicity of the preservation solution caused by adding excessive cryoprotectants to the preservation solution can also be avoided. This not only ensures the biological activity and the integrity of the original biological structure of the amniotic membrane but also further ensures higher biological safety in use.

[0019] According to the third aspect of the present invention, there is provided a method for preserving amniotic membrane, which uses the above amniotic membrane preservation solution to preserve the amniotic membrane.

[0020] Preferably, the above method for preserving amniotic membrane includes the following steps: S1. Take a healthy cesarean section placenta and bluntly dissect the amniotic membrane; S2. Rinse the taken amniotic membrane in physiological saline; S3. Lay the rinsed amniotic membrane flat on a substrate; S4. Cut it with a knife mold; S5. Place the cut amniotic membrane flat in a petri dish; S6. Add an appropriate amount of freshly prepared amniotic membrane preservation solution to submerge the amniotic membrane pieces; S7. Let it stand and soak for 4 - 10 min; S8. After soaking, take it out with forceps, bag it, and add the amniotic membrane preservation solution; S9. Seal it; S10. Perform irradiation sterilization; S11. Store the irradiated amniotic membrane at -25 to -14°C.

[0021] Preferably, in S3, the substrate includes a nitrocellulose membrane.

[0022] Preferably, in S8, the amount of amniotic membrane preservation solution added is 90 - 120 μL / m 2 . Here, "m 2 " refers to the area of the amniotic membrane.

[0023] Preferably, in S8, the amount of amniotic membrane preservation solution added is 100 μL / m 2 .

[0024] Preferably, in S10, during the irradiation sterilization process, cobalt - 60 is used for irradiation, and the irradiation dose is 5 - 40 kGy.

[0025] Preferably, in S11, the storage time is not less than 18 months.

[0026] Preferably, in S11, the storage time is 18 months.

[0027] According to the fourth aspect of the present invention, there is provided an application of the above - mentioned amniotic membrane preservation solution in preserving umbilical cord or placenta or dental tissue.

[0028] In summary, the amniotic membrane preservation solution provided by the present invention can achieve good preservation effects on the amniotic membrane under the conditions of - 25°C to - 14°C. After preservation, the morphology of the biological amniotic membrane remains unchanged, the loss rate of active factors contained is low, and at the same time, the character damage during the irradiation sterilization process of the biological amniotic membrane can be avoided. The amniotic membrane after preservation has higher biological safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 and Figure 2 are respectively the HE - stained tissue structure diagrams of the amniotic membrane after preservation in the control sample group (without irradiation) and the experimental sample group (with irradiation) in Example 2.

[0030] Figure 3 is the result of the animal experiment detection of the rat skin injury repair model for the amniotic membrane after preservation in each experimental sample group in Example 3.

[0031] Figure 4 is the schematic diagram of the state of the amniotic membrane during cryopreservation in Example 4.

[0032] Figure 5 is the result of the rabbit eye injury repair experiment for the amniotic membrane after preservation in each experimental sample group in Example 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0034] Example 1

[0035] (1) The components of the amniotic membrane preservation solution in this embodiment are as follows: 10 ml of sodium chloride injection (concentration: 0.9%), 8 ml of glycerol, 2.884 g of trehalose, and 100 mg of sodium hyaluronate.

[0036] (2) The steps for preserving the amniotic membrane using the amniotic membrane preservation solution of this embodiment are as follows:

[0037] S1. Take a healthy cesarean section placenta and bluntly dissect the amniotic membrane;

[0038] S2. Place the obtained amniotic membrane in physiological saline for rinsing;

[0039] S3. Lay the rinsed amniotic membrane flat on a nitrocellulose membrane;

[0040] S4. Cut it using a knife die;

[0041] S5. Place the cut amniotic membrane flat in a petri dish;

[0042] S6. Add an appropriate amount of freshly prepared amniotic membrane preservation solution to submerge the amniotic membrane pieces;

[0043] S7. Let it stand and soak for 5 min;

[0044] S8. After soaking, clamp it out, bag it, and add the amniotic membrane preservation solution. The dosage of the amniotic membrane preservation solution is 100 μL / m 2 ;

[0045] S9. Seal it;

[0046] S10. Sterilize it by irradiation with cobalt - 60, and the irradiation dose is 25 kGy;

[0047] S11. Place the irradiated amniotic membrane at - 20 °C for 18 months of preservation.

[0048] (3) In this embodiment, a total of two control sample groups and two experimental sample groups are set up. The numbers of the two control sample groups are 158 and 159, and the numbers of the two experimental sample groups are 202 and 204. Among them, no sodium hyaluronate is added to 158 and 159 (only different amniotic membranes are selected for 158 and 159), 100 mg of sodium hyaluronate is added to 202 and 204 (only different amniotic membranes are selected for 202 and 204). 158, 159, 202, and 204 are all preserved according to the above - mentioned preservation method, and the tensile strength of the amniotic membrane before and after preservation is tested. The specific test method is as follows: Pass an 8 - 0 wire loop, hang a 1 g weight on the wire loop, and clamp the other side with a clip and hang it on a bracket. If the amniotic membrane does not break within 5 min, the elasticity test is qualified.

[0049] The above - mentioned test results are shown in Table 1.

[0050] Tensile strength test results before and after the preservation of 158, 159, 202, and 204 in Table 1

[0051]

[0052] As can be seen from Table 1, when using the preservation solution provided by the present invention to preserve fresh amniotic membrane, it will not affect the humidity and flexibility of the amniotic membrane itself. The preserved amniotic membrane still has the same mechanical strength as the fresh amniotic membrane. Therefore, the subsequent further use of the amniotic membrane can be realized.

[0053] Example 2

[0054] (1) The composition of the amniotic membrane preservation solution in this example is as follows: 12 ml of sodium chloride injection (concentration 0.9%), 8 ml of glycerol, 2.884 g of trehalose, and 100 mg of sodium hyaluronate.

[0055] (2) The steps for preserving the amniotic membrane using the amniotic membrane preservation solution of this example are the same as those in Example 1.

[0056] (3) In this example, a control sample group and an experimental sample group were set up. The control sample group did not perform the irradiation operation in (2) (i.e., did not perform S10), and the experimental sample group preserved the amniotic membrane completely according to the steps in (2). The preserved amniotic membranes in these two sample groups were respectively subjected to HE staining detection, and the results are as Figure 1 and Figure 2 shown. It can be seen that before irradiation, the amniotic membrane tissue structure was intact ( Figure 1 ), and after irradiation with a cobalt-60 radiation source at an irradiation dose of 25 kGy, the amniotic membrane tissue structure was also intact. Therefore, in addition to retaining the natural structural integrity of the amniotic membrane tissue, the amniotic membrane in this example can also ensure the aseptic requirements of the final product, wet biological amniotic membrane, and improve the clinical use of the wet biological amniotic membrane as a medical device.

[0057] Example 3

[0058] (1) In this example, 4 experimental sample groups were set up, numbered 167, 172, 174, and 202 respectively. Among them, 202 represents that it is the same as the 202 experimental sample group in Example 1 (i.e., the treatment method is the same); while 167, 172, and 174 all used the following composition of the amniotic membrane preservation solution for preservation treatment: 24 ml of sodium chloride injection (concentration 0.9%), 16 ml of glycerol, 5.768 g of trehalose, and 100 mg of sodium hyaluronate, and the irradiation dose in S10 was adjusted to 8.8 kGy. Only the specific amniotic membranes selected were different, and the other treatment steps were the same as those in (2) of Example 1.

[0059] (2) The total protein, TGF-β1, EGF, bFGF, KGF, and NGF contents of the amniotic membranes before and after preservation in the 4 experimental samples numbered 167, 172, 174, and 206 were tested respectively. The specific testing method is as follows: Take an amniotic membrane of 10×10 mm 2 in size and add 200 μL of RIPA lysis buffer. Grind it into a slurry with a tissue grinder, place it on ice for lysis for 30 min, then centrifuge at 12,000 rpm for 5 min to take the supernatant, which is the test solution. The test solution was detected according to the instructions of the BCA protein concentration assay kit (enhanced type) and ELISA kit.

[0060] The above test results are shown in Table 2.

[0061] Table 2 Test results of the active ingredient contents of the amniotic membranes in each experimental sample group of 167, 172, 174, and 206 before and after preservation

[0062]

[0063] As can be seen from Table 2, after the amniotic membrane is preserved for a certain period of time using the amniotic membrane preservation solution provided by the present invention, various natural active ingredients of the amniotic membrane can still be effectively retained, including collagen, growth factors, anti-inflammatory factors, etc., and it has the functions of anti-inflammation, anti-angiogenesis, anti-scarring, and promoting healing of fresh amniotic membranes. Further comparing the data in Table 2, it can be seen that the contents of various natural active ingredients of the biological amniotic membrane preserved using the amniotic membrane preservation solution of the present invention are less different from those of fresh amniotic membranes, indicating that the amniotic membrane preservation solution and preservation method provided by the present invention can effectively retain various natural components of the amniotic membrane.

[0064] Furthermore, in this embodiment, the amniotic membranes after preservation in 167, 172, and 174 were also used for animal experiments on the rat skin injury repair model. The specific experimental process was as follows: A 1 cm 2 circular full-thickness skin defect model was created on the back of the rat. The amniotic membrane was applied or not applied according to the experimental grouping, and an elastic bandage was covered. Photos were taken on DAY0, DAY3, and DAY7 to observe the wound healing situation.

[0065] The results of the animal experiments on the rat skin injury repair model are as Figure 3 shown. The results show that the postoperative treatment effect is significant, no vascularized scar formation, no inflammation and no rejection reaction, and it has an excellent therapeutic effect.

[0066] It should be noted here that in Figure 3 , the rat wound treatments were irradiated amniotic membrane, non-irradiated amniotic membrane, and blank (without using amniotic membrane treatment) from top to bottom. The results show that the irradiated amniotic membrane during the preservation process has the best wound repair effect.

[0067] Example 4

[0068] (1) The components of the amniotic membrane preservation solution in this embodiment are as follows: 10 ml of sodium chloride injection (concentration 0.9%), 8 ml of glycerol, 1.368 g of trehalose, and 100 mg of sodium hyaluronate.

[0069] (2) The steps for preserving the amniotic membrane using the amniotic membrane preservation solution of this embodiment are the same as those in Example 1.

[0070] (3) By observing the amniotic membrane preserved in this embodiment at -20°C, as Figure 4 shown, it shows that the amniotic membrane will not freeze during storage, which can effectively reduce the damage of ice crystals to the amniotic membrane, maintain the wet environment of the amniotic membrane, and thus is beneficial to maintaining the biological activity and original biological structure of the amniotic membrane.

[0071] Example 5

[0072] (1) A total of 3 experimental sample groups and a control sample group (non-treatment group) were set up in this embodiment, numbered 190, 204, 202, and non-treatment group respectively. Among them, 204 and 202 represent that they are the same as the 204 and 202 experimental sample groups in Example 1 (i.e., the treatment methods are the same); while 190 was preserved and treated with the following components of the amniotic membrane preservation solution: 10 ml of sodium chloride injection (concentration 0.9%), 10 ml of glycerol, 0.684 g of trehalose, and 100 mg of sodium hyaluronate. Only the specific amniotic membrane selected was different, and the other treatment steps were the same as those in (2) of Example 1; the non-treatment group was not treated with the amniotic membrane.

[0073] (2) Rabbit eye injury repair experiments were respectively carried out on the amniotic membranes before and after preservation in the 4 experimental samples numbered 190, 204, 202, and non-treatment group. The specific experimental process is as follows: The rabbit cornea was drilled with a trephine with a diameter of 1 cm and positioned as the injury site. The corneal epithelium was cut off with a lateral cutting knife. After the cornea was rinsed with physiological saline, the amniotic membrane was covered on the cornea and sutured to the sclera to fix it on the cornea. The control group only performed the suture operation. All rabbits were put on a net-like elastic bandage on the head and wore an Elizabethan collar to prevent the rabbits from scratching. The sutures of the modeled eyes were removed at time points, and slit lamp photographs were taken.

[0074] The results of the rabbit eye injury repair experiment are as Figure 5 shown. The results show that the amniotic membrane preserved using the amniotic membrane preservation solution provided by the present invention has excellent repair effects, and the repair effect in the 202 experimental group is the best.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. An amniotic membrane preservation solution, characterized in that: It includes the following components: sodium chloride injection, glycerol, trehalose, sodium hyaluronate; The volume ratio of the sodium chloride injection to the glycerol is 8-24:8-18; The volume mass ratio of the sodium chloride injection to trehalose is 8-24:0.5-6; The mass ratio of the trehalose to the sodium hyaluronate is 0.5-6:0.006-0.

15.

2. The amniotic membrane preservation solution according to claim 1, characterized in that: The volume ratio of the sodium chloride injection to the glycerol is 10:10; The volume mass ratio of the sodium chloride injection to trehalose is 10:0.684; The mass ratio of the trehalose to the sodium hyaluronate is 0.684:0.

1.

3. Use of the amniotic membrane preservation solution according to any one of claims 1 to 2 for preserving amniotic membrane.

4. The use of the amniotic membrane preservation solution for preserving amniotic membrane as claimed in claim 3, characterized in that: During the storage of the amniotic membrane, the temperature is -25 to -14°C.

5. A method for preserving amniotic membrane, characterized in that: The amniotic membrane is preserved using the amniotic membrane preservation solution according to any one of claims 1 to 2.

6. The method for preserving amniotic membrane according to claim 5, characterized in that: The steps include: S1. Take the healthy cesarean section placenta and bluntly remove the amniotic membrane; S2. The amniotic membrane is placed in saline for washing; S3. Spreading the washed amniotic membrane on a substrate; S4. Use die cutting to cut; S5. Place the cut amniotic membrane flat in a culture dish; S6. Add an appropriate amount of the freshly prepared amniotic membrane preservation solution to cover the amniotic membrane; S7. Let it soak for 4 to 10 minutes; S8. After soaking, remove the membrane, bag it, and add the amniotic membrane preservation solution; S9. Sealing; S10. Irradiation sterilization; S11. Store the irradiated amniotic membrane at -25 to -14°C.

7. The method for preserving amniotic membrane according to claim 6, characterized in that: In S3, the substrate includes a nitrocellulose membrane.

8. The method for preserving amniotic membrane according to claim 6, characterized in that: In S8, the amount of the amniotic fluid added is 100 μL / m 2 .

9. The method for preserving amniotic membrane according to claim 6, characterized in that: In the S10, during the irradiation sterilization process, cobalt-60 is used for irradiation, and the irradiation dose is 5 to 40 kGy.

10. Use of the amniotic membrane preservation solution according to claims 1 to 2 for preserving umbilical cord, placenta or tooth tissue.

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