An organ preservation solution for ovary and its preparation method and application

Abstract

The application relates to an organ preservation solution for an ovary and a preparation method and application thereof, the preservation solution comprising a basic component and an additive component, the basic component comprising a basic culture solution and a buffer component, and the additive component comprising vitamin B12 and melatonin. The organ preservation solution has good cryopreservation effect on the ovary, provides a suitable osmotic pressure, an acid-base environment and sufficient nutrients, improves the antioxidation, thereby prolongs the static refrigeration time of the ovary, improves the survival rate of cells in the ovary, and makes up for the defects of the current non-ovarian special cryopreservation solution.

Description

Technical Field

[0001] This invention belongs to the field of material preservation technology, and relates to an organ preservation solution for ovaries, its preparation method and application. Background Technology

[0002] With the development of tumor diagnosis and treatment technologies, the number of women of childbearing age or pre-puberty who survive cancer is gradually expanding. These patients may experience impaired or even lost fertility due to the cancer itself or cancer treatments (chemotherapy, radiotherapy, surgery). Therefore, developing effective and safe fertility preservation technologies is crucial. The aim is to preserve gonadal tissue or cells before patients undergo reproductive toxicity treatments to maintain fertility; and after the patient completes treatment and meets the basic conditions for fertility, the gonadal tissue or cells can be re-transplanted back into the patient to restore reproductive and endocrine functions. Ovarian cryopreservation and autologous transplantation is currently the only available method of fertility preservation for patients with contraindications to hormone use, pre-puberty patients, or those without sufficient time for ovulation induction. After ovarian retrieval, it is necessary to promptly preserve the ovary in a low-temperature environment and transport it to the laboratory for subsequent cryopreservation. The liquid used in this cryopreservation and transportation is an ovarian preservation solution, which needs to maintain the viability of the isolated ovary, protect its function, and reduce damage from low temperature and ischemia-hypoxia.

[0003] The corresponding organ preservation solutions used in other organ transplants, such as those for the liver, heart, and kidneys (UW solution, HTK solution, Celsior solution, HCA solution, etc.), are designed based on the cell type, osmotic pressure, pH, ion balance inside and outside the cell membrane, and metabolic characteristics of the organ. For example, UW solution uses macromolecules to maintain osmotic pressure and adds glutathione and allopurinol to fight free radicals; it is used as a preservation solution for liver, pancreas and kidney transplantation. HTK solution uses histidine as a buffer system, adds tryptophan and ketoglutarate to promote ATP production, has low potassium to reduce calcium overload, and adds glycine, alanine and LK614 to reduce oxygen free radical damage. Celsior solution uses lacturonic acid as an osmotic pressure component and histidine as a buffer system; it is a high-sodium, low-potassium extracellular fluid type preservation solution. Low potassium restricts the opening of voltage-dependent calcium channels, restricts calcium ion entry into cells, and avoids depolarization of smooth muscle cell membranes; it is suitable for cardiac smooth muscle and is used as a preservation solution for heart transplantation. HCA solution is a hypertonic citrate purine solution, high potassium and low sodium (intracellular fluid type); it uses histidine as a buffer system, dextran to maintain osmotic pressure, and adds ligustrazine to remove oxygen free radicals and avoid calcium overload; it is used as a preservation solution for kidney transplantation.

[0004] Currently, there are no commercially available organ preservation solutions specifically designed for the ovaries. Preservation solutions for organs such as the liver, kidneys, heart, and pancreas are not suitable for the ovaries in terms of osmotic pressure, intracellular and extracellular ion balance, and pH. For example, when placed in a hypertonic solution transported by the kidneys, oocytes in the ovary will rapidly shrink and degenerate. Ovarian preservation solutions used in scientific research are mostly room-temperature or 37°C operating solutions used for handling oocytes or embryos outside of incubators. These provide the ovaries with basic nutrients and suitable osmotic pressure, but cannot mitigate the hypothermia, ischemia, and hypoxia damage faced by the ovaries during cryogenic transport. To achieve efficient preservation of gonadal function, there is an urgent clinical need for organ preservation solutions specifically designed for the ovaries that can improve the quality of ovarian cryopreservation. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an organ preservation solution for the ovary, its preparation method and application.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an organ preservation solution for the ovary, the preservation solution comprising a base component and an additive component, the base component comprising a base culture medium and a buffer component, and the additive component comprising vitamin B12 and melatonin.

[0008] Organ preservation solutions need to be designed based on factors such as cell type, suitable osmotic pressure, pH, ion balance across the cell membrane, and metabolic characteristics. For example, hypertonic solutions are used for renal transport, while high-sodium, low-potassium preservation solutions are used for the heart. Currently, there are no commercially available cryopreservation solutions suitable for the ovaries. Preservation solutions used for other organs are not suitable for the ovaries in terms of osmotic pressure, pH, and ion balance across the cell membrane. For instance, placing oocytes in a hypertonic solution used for renal transport would cause them to rapidly shrink and degenerate.

[0009] The ovarian or oocyte cryopreservation solutions used in scientific research or existing patents are not the same type of product as the organ preservation solution of this invention. Cryopreservation solutions are used for cryopreservation at extremely low temperatures (such as liquid nitrogen, -196°C), replacing water from cells to reduce damage from ice crystal formation during the cooling process. The organ preservation solution of this invention is used for cryogenic (0-4°C) transport of ovaries, aiming to reduce the hypothermia, ischemia, and hypoxia damage faced by the ovaries. The required osmotic pressure, temperature, composition, and corresponding anti-damage mechanisms are completely different. If the ovarian cryopreservation solution is directly applied to the cryogenic transport process, it will cause dehydration of the ovarian tissue and oocytes, resulting in loss of activity during subsequent mechanical manipulation. The ovarian cryopreservation solutions used in scientific research are mostly operating solutions originally designed for handling oocytes or embryos at room temperature or 37°C outside the incubator, providing basic nutrients and suitable osmotic pressure for the ovaries, but failing to mitigate the hypothermia, ischemia, and hypoxia damage faced by the ovaries during cryogenic transport.

[0010] This organ preservation solution exhibits excellent cryopreservation effects on ovaries, providing suitable osmotic pressure, pH environment, and sufficient nutrients to enhance antioxidant capacity. This prolongs the static cryopreservation time of the ovaries and increases the cell survival rate, overcoming the current deficiency of a dedicated cryopreservation solution for ovaries. This invention holds promise for providing effective cryopreservation and transport technology support for patients who need to preserve their fertility through ovarian cryopreservation, and has excellent application prospects.

[0011] Preferably, the preservation solution comprises, in molar concentration, 1-20 nM vitamin B12 and 0.02-0.2 mM melatonin.

[0012] The molar concentration of vitamin B12 in the preservation solution can be selected from 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, etc., and the molar concentration of melatonin can be selected from 0.02 mM, 0.03 mM, 0.0 4mM, 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.1mM, 0.11mM, 0.12mM, 0.13mM, 0.14mM, 0.15mM, 0.16mM, 0.17mM, 0.18mM, 0.19mM, 0.2mM, etc. Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0013] The amount of vitamin B12 and melatonin added is very important for preservation.

[0014] Preferably, the buffering component includes any one or a combination of at least two of the following: potassium dihydrogen phosphate-disodium hydrogen phosphate buffer pair, histidine-histidine hydrochloride buffer pair, or HEPES.

[0015] Preferably, the preservation solution comprises, in molar concentrations, 0.1-0.6 mM potassium dihydrogen phosphate and 0.1-0.5 mM disodium hydrogen phosphate.

[0016] The molar concentration of potassium dihydrogen phosphate can be selected from 0.1mM, 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5mM, 0.55mM, 0.6mM, etc., and the molar concentration of disodium hydrogen phosphate can be selected from 0.1mM, 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5mM, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0017] Preferably, the preservation solution comprises 50-150 mM histidine and 5-15 mM histidine hydrochloride in molar concentration.

[0018] The molar concentration of histidine can be selected from 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, etc., and the molar concentration of histidine hydrochloride can be selected from 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0019] Preferably, the preservation solution includes HEPES 10-50mM in molar concentration, such as 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, etc. Other specific values ​​within the above range can be selected, and will not be described in detail here.

[0020] Preferably, the basic culture medium includes M199 culture medium or HTF culture medium.

[0021] Preferably, the pH of the organ preservation solution for the ovary is 7.2-7.4, such as 7.2, 7.3, 7.4, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0022] The pH values ​​were measured at 30-35℃.

[0023] Preferably, the osmotic pressure of the organ preservation solution for the ovary is 270-300 m0sm / kg, such as 270 m0sm / kg, 275 m0sm / kg, 280 m0sm / kg, 285 m0sm / kg, 290 m0sm / kg, 295 m0sm / kg, 300 m0sm / kg, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0024] In a second aspect, the present invention provides a method for preparing an organ preservation solution for the ovary according to the first aspect, the method comprising: physically mixing the base components and the additive components before use, thereby obtaining the solution.

[0025] Thirdly, the present invention provides the application of the organ preservation solution for ovaries according to the first aspect in the cryopreservation of ovaries.

[0026] Preferably, the temperature for ovarian preservation is 0-4℃, such as 0℃, 0.5℃, 1℃, 1.5℃, 2℃, 2.5℃, 3℃, 3.5℃, 4℃, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0027] Preferably, the specific steps of the application include: mixing the organ preservation solution for the ovary with an HSA solution or an SPS solution, and then placing the isolated ovary in the solution.

[0028] Preferably, the mass percentage of HSA in the HSA solution is 10-20%, such as 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0029] Preferably, the mass percentage of SPS in the SPS solution is 10-20%, such as 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This organ preservation solution exhibits excellent cryopreservation effects on ovaries, providing suitable osmotic pressure, pH environment, and sufficient nutrients to enhance antioxidant capacity. This prolongs the static cryopreservation time of the ovaries and increases the cell survival rate, overcoming the current deficiency of a dedicated cryopreservation solution for ovaries. This invention holds promise for providing effective cryopreservation and transport technology support for patients who need to preserve their fertility through ovarian cryopreservation, and has excellent application prospects. Detailed Implementation

[0032] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0033] Example 1

[0034] This embodiment provides an organ preservation solution for the ovary, comprising the following components: vitamin B12 2nM, melatonin 0.1mM, potassium dihydrogen phosphate 0.44mM, disodium hydrogen phosphate 0.336mM, histidine 90mM, histidine hydrochloride 9mM, and HEPES 25mM, with M199 culture medium as the dissolving medium.

[0035] The preparation method is as follows: before use, the above raw materials are physically mixed to obtain the product.

[0036] Example 2

[0037] This embodiment provides an organ preservation solution for the ovary, comprising the following components: vitamin B12 1 nM, melatonin 0.2 mM, potassium dihydrogen phosphate 0.1 mM, disodium hydrogen phosphate 0.1 mM, histidine 50 mM, histidine hydrochloride 5 mM, and HEPES 50 mM, with M199 culture medium as the dissolving medium.

[0038] The preparation method is the same as in Example 1.

[0039] Example 3

[0040] This embodiment provides an organ preservation solution for the ovary, comprising the following components: vitamin B12 20 nM, melatonin 0.02 mM, potassium dihydrogen phosphate 0.6 mM, disodium hydrogen phosphate 0.5 mM, histidine 150 mM, histidine hydrochloride 15 mM, and HEPES 10 mM, with M199 culture medium as the dissolving medium.

[0041] The preparation method is the same as in Example 1.

[0042] Comparative Example 1

[0043] This comparative example provides an organ preservation solution for the ovary, which differs from Example 1 only in that it does not contain vitamin B12, while the other components and contents remain unchanged.

[0044] The preparation method is the same as in Example 1.

[0045] Comparative Example 2

[0046] This comparative example provides an organ preservation solution for the ovary, which differs from Example 1 only in that it does not contain melatonin, while the other components and contents remain unchanged.

[0047] The preparation method is the same as in Example 1.

[0048] Comparative Example 3

[0049] This comparative example provides an organ preservation solution for the ovary, which differs from Example 1 only in that it does not contain vitamin B12 and melatonin, while the other ingredients and contents remain unchanged.

[0050] The preparation method is the same as in Example 1.

[0051] Comparative Example 4

[0052] This comparative example provides an organ preservation solution for the ovary, which differs from Example 1 only in that it does not contain vitamin B12, and the reduced mass of vitamin B12 is allocated to the mass of melatonin, while other components and contents remain unchanged.

[0053] The preparation method is the same as in Example 1.

[0054] Comparative Example 5

[0055] This comparative example provides an organ preservation solution for the ovary, which differs from Example 1 only in that it does not contain melatonin and replaces "vitamin B12 2 nM" with "vitamin B12 10 nM", while other ingredients and contents remain unchanged.

[0056] The preparation method is the same as in Example 1.

[0057] Test Example 1

[0058] Test samples: Examples 1-3, Comparative Examples 1-5

[0059] Test method:

[0060] Bovine ovaries were preserved at 4°C for 24 hours after the addition of 10% HSA solution, with 5 ovaries per group. After 24 hours, thin sections of ovarian cortex (1cm×1cm in area, 1mm in thickness) were prepared. The cortical sections were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The proportion of normal follicles (number of normal follicles / total number of follicles) was calculated. At the same time, a fresh cortical fragment of approximately 2mm*2mm*1mm in size was collected from each ovary and analyzed by flow cytometry using the Annexin V-FITC apoptosis detection kit to calculate the apoptosis rate of the ovarian tissue fragment.

[0061] Table 1

[0062] Example 1 487 87.1％(424 / 487) 14.35±2.3 Example 2 472 85.8％(405 / 472) 16.27±1.5 Example 3 489 85.7％(419 / 489) 16.01±2.1 Comparative Example 1 550 80％(440 / 550) 18.36±2.7 Comparative Example 2 456 80.9％(369 / 456) 18.69±3.2 Comparative Example 3 502 70.1％(352 / 502) 25.46±5.6 Comparative Example 4 449 79.5％(357 / 449) 19.12±2.8 Comparative Example 5 452 80.8％(365 / 452) 19.01±1.6

[0063] The proportion of normal morphological follicles in the example group was higher than that in the control group, and the apoptosis rate was lower in the example group than in the control group, with statistically significant differences. The proportion of normal morphological follicles in the group with combined vitamin B12 and melatonin (example group) was higher than that in the group with melatonin alone (control group 1) and the group with vitamin B12 alone (control group 2), and the apoptosis rate was lower in the group with vitamin B12 alone and the group with melatonin alone, with statistically significant differences. This demonstrates that vitamin B12 and melatonin can work synergistically to achieve the above effects.

[0064] When melatonin was added alone, even if the reduced mass of vitamin B12 was allocated to melatonin (Comparative Example 4), the effect of the Example Group could not be achieved. The proportion of normal morphological follicles was lower and the apoptosis rate was higher than that of the Example Group. When vitamin B12 was added alone, even if a portion of the reduced mass of melatonin was allocated to vitamin B12 under appropriate vitamin B12 concentration control (Comparative Example 5), the effect of the Example Group could not be achieved. The proportion of normal morphological follicles was lower and the apoptosis rate was higher than that of the Example Group.

[0065] Organ preservation solutions containing added vitamin B12 and melatonin can improve the survival rate of cells in ovarian cells transported at low temperatures and improve the quality of ovarian cryopreservation. The combined addition of vitamin B12 and melatonin is more effective than adding vitamin B12 or melatonin alone, and the amount of each is very important for the above effects.

[0066] The applicant declares that this invention illustrates an organ preservation solution for the ovary, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0067] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0068] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. An organ preservation solution for ovaries, characterized in that, The preservation solution includes basic components and additives. The basic components include basic culture medium and buffer components, and the additives are vitamin B12 and melatonin. The preservation solution comprises, in molar concentrations, 1-20 nM vitamin B12 and 0.02-0.2 mM melatonin; The buffering components include any one or a combination of at least two of the following: potassium dihydrogen phosphate-disodium hydrogen phosphate buffer pair, histidine-histidine hydrochloride buffer pair, or HEPES. The basic culture medium includes M199 culture medium or HTF culture medium.

2. The organ preservation solution for the ovary according to claim 1, characterized in that, The preservation solution comprises, in molar concentrations, 0.1-0.6 mM potassium dihydrogen phosphate and 0.1-0.5 mM disodium hydrogen phosphate.

3. The organ preservation solution for the ovary according to claim 1, characterized in that, The preservation solution comprises, in molar concentrations, 50-150 mM histidine and 5-15 mM histidine hydrochloride.

4. The organ preservation solution for the ovary according to claim 1, characterized in that, The preservation solution contains 10-50 mM HEPES in molar concentration.

5. The organ preservation solution for the ovary according to claim 1, characterized in that, The pH of the organ preservation solution used for the ovary is 7.2-7.

4.

6. A method for preparing an organ preservation solution for the ovary according to any one of claims 1-5, characterized in that, The preparation method includes: physically mixing the base component and the additive component before use, thereby obtaining the product.

7. The use of an organ preservation solution for ovaries according to any one of claims 1-5 in the cryopreservation of ovaries.

8. The application according to claim 7, characterized in that, The ovaries were preserved at a temperature of 0-4℃.

9. The application according to claim 7 or 8, characterized in that, The specific steps of the application include: mixing the organ preservation solution for the ovary with an HSA solution or an SPS solution, and then placing the isolated ovary in the solution.