A preservation solution for aborted tissue, its preparation method and application

Abstract

The present invention relates to a tissue preservation solution for aborted tissues, characterized in that the tissue preservation solution for aborted tissues comprises PBS and a tissue preservation solution; preferably, the tissue preservation solution for aborted tissues is a mixed solution of PBS and the tissue preservation solution. The present invention provides for the first time a tissue preservation solution that can stably preserve and transport aborted tissues at room temperature, reducing the temperature requirements during the preservation and transportation of aborted tissues, thereby achieving the purpose of saving preservation costs and reducing transportation costs. At the same time, through the research on the proportioning of the tissue preservation solution, the proportion of the expensive tissue preservation solution in the tissue preservation solution for aborted tissues of the present invention is reduced, and the cost of the tissue preservation solution for aborted tissues is lowered.

Description

Technical Field

[0001] The present invention relates to the field of tissue preservation, and particularly to a tissue preservation solution for aborted products, a preparation method thereof, and an application thereof. Background Art

[0002] Abortion is the most common complication during early pregnancy. Clinically, about 15 - 25% of pregnancies end in abortion, and most occur in the first trimester. Although there are many known causes and risk factors for early pregnancy abortion, approximately 60% of cases are caused by chromosomal abnormalities. These cytogenetic abnormalities include autosomal trisomy (27%), polyploidy (10%), X chromosome monosomy (9%), and structural rearrangements (2%); ditrisomy and multitrisomy are not common, with an incidence of approximately 0.7%. Recurrent pregnancy loss (RPL) is defined as two or more consecutive spontaneous abortions. According to statistics, less than 5% of women experience two consecutive abortions, and only 1% of women experience three or more. In more than 50% of RPL couples, the disease has unexplained or idiopathic causes. One possible reason is that these couples have an increased tendency to produce aneuploid embryos, which then lead to more abortions. Marquard et al. (in 2010) found that 80% of abortions occur in women over 35 years old, and without chromosomal testing of aborted tissue, the causes of many RPL cases cannot be explained.

[0003] In medical research and clinical applications, biological samples are the bridge connecting molecular information and diseases. According to the difference in usage purposes, the preservation requirements of samples also vary. Samples used for gene detection extraction need to have their molecular information well-preserved without degradation during the preservation process. Aborted tissue refers to the embryonic tissue and other related tissues discharged from the endometrium during natural or induced abortion, such as amniotic sacs, placentas, umbilical cords, and trophoblastic tissues. These tissues are sent to the laboratory for pathological examination or gene detection to determine whether the pregnancy is developing normally or whether the embryo has a genetic disease caused by gene mutations, resulting in abortion. However, the aborted tissue of some patients is not detected locally and needs to be preserved and transported to other places for professional testing. Therefore, the accuracy and effectiveness of laboratory test results are affected not only by factors such as experimental methods, detection reagents, and experimental operations but also by the preservation and transportation links of the samples. A good tissue preservation solution for aborted products can keep the intact biological information of the samples during preservation and transportation.

[0004] Currently, the preservation of abortus tissue samples in the industry is basically carried out using pure PBS buffer or pure tissue preservation solution. However, when only PBS buffer is used for preservation, the sample tissue is prone to degradation and is only suitable for short-term temporary preservation; if only the tissue preservation solution is used for preservation, although the sample tissue is not easily degraded, it is expensive. In addition, both the preservation and transportation of tissue samples require freezing conditions, which greatly increases the transportation cost. Summary of the Invention

[0005] Problems to be Solved by the Invention

[0006] In order to solve the above problems existing in the prior art, the present invention provides an abortus tissue preservation solution, enabling the abortus tissue to be stably preserved in an economical and applicable tissue preservation solution at room temperature and successfully transported for several days, thereby achieving the purpose of saving preservation costs and reducing transportation costs.

[0007] Solutions for Solving the Problems

[0008] The present invention provides an abortus tissue preservation solution, characterized in that the abortus tissue preservation solution comprises PBS and tissue preservation solution.

[0009] Preferably, the abortus tissue preservation solution is a mixed solution of PBS and tissue preservation solution.

[0010] Preferably, the ratio of PBS to tissue preservation solution is 15:1 - 1:1; more preferably, the ratio of PBS to tissue preservation solution is 9:1.

[0011] Preferably, the tissue preservation solution comprises DMSO and EDTA.

[0012] More preferably, the tissue preservation solution is a mixed solution of DMSO and EDTA.

[0013] Preferably, the ratio of DMSO to EDTA is 1:1 - 1:10; more preferably, the ratio of DMSO to EDTA is 1:4.

[0014] The present invention also provides a preparation method of the abortus tissue preservation solution, characterized in that the preparation method comprises the following steps:

[0015] (1) Mix DMSO and EDTA evenly to obtain the tissue preservation solution;

[0016] (2) Mix PBS and the tissue preservation solution obtained in step (1) evenly to obtain the abortus tissue preservation solution.

[0017] Preferably, in step (1), the ratio of DMSO to EDTA is 1:1 - 1:10; more preferably, the ratio of DMSO to EDTA is 1:4.

[0018] Preferably, the ratio of PBS to the tissue preservation solution in step (2) is 15:1 - 1:1; more preferably, the ratio of PBS to the tissue preservation solution is 9:1.

[0019] The present invention also provides an application of the aborted tissue preservation solution or the aborted tissue preservation solution prepared according to the preparation method in the preservation and transportation of tissue samples.

[0020] Preferably, the preservation is to completely immerse the aborted tissue after ex vivo in the aborted tissue preservation solution and preserve it at room temperature.

[0021] Effects of the Invention

[0022] The present invention for the first time provides a tissue preservation solution that can stably preserve and transport aborted tissue at normal temperature, reduces the temperature requirements during the preservation and transportation of aborted tissue, thereby achieving the purpose of saving preservation costs and reducing transportation costs.

[0023] Meanwhile, through the research on the formulation of the tissue preservation solution, the proportion of expensive tissue preservation solution in the aborted tissue preservation solution of the present invention is reduced, and the cost of the aborted tissue preservation solution is lowered. Description of the Drawings

[0024] Figure 1 It is the agarose gel electrophoresis detection pattern of the sample with a preservation time of 3 days.

[0025] Figure 2 It is the agarose gel electrophoresis detection pattern of the sample with a preservation time of 5 days. Detailed Embodiments

[0026] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following will be described in detail by listing specific embodiments. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical and scientific fields to which this application belongs.

[0027] The term "recurrent pregnancy loss (RPL)" used in the present invention means two or more consecutive spontaneous abortions.

[0028] The term "PBS" used in the present invention means phosphate buffered saline, which has functions such as maintaining the stability of the tissue pH value, preventing the tissue from acid-base balance disorders, preventing cell membrane rupture and cell lysis, etc., and helps to maintain the integrity of tissue structure and function. In addition, PBS can also provide the ions and nutrients required for cell metabolism and delay the cell death rate.

[0029] The term "DMSO" used in the present invention refers to dimethyl sulfoxide, which has good permeability and antioxidant effects, can promote protein folding and stability, maintain enzyme activity, reduce the damage of ice crystals to cells and tissues, delay the aging process of cells and tissues, and help maintain the biological characteristics of tissues.

[0030] The term "EDTA" used in the present invention refers to ethylenediaminetetraacetic acid, which can chelate ions to combine with heavy metal ions such as calcium and magnesium to form complexes, preventing these ions from reacting and oxidizing with cell membranes, nucleic acids and proteins, thereby reducing cell damage and death. In addition, EDTA can also inhibit the activity of some enzymes, thereby delaying the cell metabolism process and helping to maintain the stability of cell structure and function.

[0031] Unless otherwise specified, the experimental methods without specific conditions in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions, or parts are by weight. Unless otherwise specified, the room temperature described in the present invention is 20°C to 30°C. Unless otherwise specified, the various materials and reagents used in the present invention can be obtained by conventional methods in the art or through commercial channels.

[0032] The present invention provides a tissue preservation solution for miscarriage products, characterized in that the tissue preservation solution for miscarriage products comprises PBS and a tissue preservation solution.

[0033] In certain embodiments, the tissue preservation solution for miscarriage products is a mixed solution of PBS and a tissue preservation solution.

[0034] In certain embodiments, the ratio of PBS to the tissue preservation solution is 15:1 - 1:1.

[0035] In certain embodiments, the ratio of PBS to the tissue preservation solution is 9:1.

[0036] In certain embodiments, the tissue preservation solution comprises DMSO and EDTA.

[0037] In certain embodiments, the tissue preservation solution is a mixed solution of DMSO and EDTA.

[0038] In certain embodiments, the ratio of DMSO to EDTA is 1:1 - 1:10.

[0039] In certain embodiments, the ratio of DMSO to EDTA is 1:4.

[0040] In certain embodiments, the tissue preservation solution for miscarriage products can preserve the miscarriage product tissue at room temperature for 0 - 7 days.

[0041] In certain embodiments, the aborted tissue preservation solution can preserve aborted tissue at room temperature for 0 - 5 days.

[0042] In certain embodiments, the aborted tissue preservation solution can preserve aborted tissue at room temperature for 3 - 5 days.

[0043] The present invention also provides a method for preparing the aborted tissue preservation solution, which is characterized in that the preparation method comprises the following steps:

[0044] (1) Mix DMSO and EDTA evenly to obtain a tissue preservation solution;

[0045] (2) Mix PBS and the tissue preservation solution obtained in step (1) evenly to obtain the aborted tissue preservation solution.

[0046] In certain embodiments, the addition order of each component can effectively improve the anti - pollution ability of the aborted tissue preservation solution, and ensure that each component can be fully mixed evenly, better playing a synergistic role.

[0047] In certain embodiments, the ratio of DMSO to EDTA in step (1) is 1:1 - 1:10.

[0048] In certain embodiments, the ratio of DMSO to EDTA in step (1) is 1:4.

[0049] In certain embodiments, the ratio of PBS to the tissue preservation solution in step (2) is 15:1 - 1:1.

[0050] In certain embodiments, the ratio of PBS to the tissue preservation solution in step (2) is 9:1.

[0051] In certain embodiments, based on 1 L of the aborted tissue preservation solution, the dosage of PBS is 0 - 1000 mL.

[0052] In certain embodiments, based on 1 L of the aborted tissue preservation solution, the dosage of PBS is 500 - 900 mL.

[0053] In certain embodiments, the role of PBS is to maintain the stability of tissue pH value, prevent tissue from acid - base balance disorder, and at the same time prevent cell membrane rupture and cell lysis, contributing to maintaining the integrity of tissue structure and function.

[0054] In certain embodiments, the role of PBS can also be to provide ions and nutrients required for cell metabolism and delay the cell death rate.

[0055] In certain embodiments, based on 1 L of the aborted tissue preservation solution, the dosage of DMSO is 0 - 200 mL.

[0056] In certain embodiments, based on 1 L of the aborted tissue preservation solution, the dosage of DMSO is 20 - 100 mL.

[0057] In certain embodiments, DMSO has good permeability and antioxidant effects, can promote protein folding and stability, maintain enzyme activity, reduce the damage of ice crystals to cells and tissues, delay the aging process of cells and tissues, and is beneficial to maintaining the biological characteristics of tissues.

[0058] In certain embodiments, based on 1 L of the aborted tissue preservation solution, the dosage of EDTA is 0 - 800 mL.

[0059] In certain embodiments, based on 1 L of the aborted tissue preservation solution, the dosage of EDTA is 80 - 400 mL.

[0060] In certain embodiments, EDTA can chelate ions to combine with heavy metal ions such as calcium and magnesium to form complexes, preventing these ions from reacting and oxidizing with cell membranes, nucleic acids and proteins, thereby reducing cell damage and death.

[0061] In certain embodiments, EDTA can also inhibit the activity of some enzymes, delay the cell metabolism process, and is beneficial to maintaining the stability of cell structure and function.

[0062] The present invention also provides an application of the aborted tissue preservation solution or the aborted tissue preservation solution prepared according to the preparation method in the preservation and transportation of tissue samples.

[0063] In certain embodiments, the preservation is to completely immerse the aborted tissue after excision in the aborted tissue preservation solution and store it at room temperature.

[0064] In certain embodiments, when using the aborted tissue preservation solution to preserve the aborted tissue, the aborted tissue after excision is completely immersed in the aborted tissue preservation solution and stored at 25°C.

[0065] Example 1: Preparation of the aborted tissue preservation solution

[0066] Mix the DMSO and EDTA solutions evenly in a ratio of 1:4 to obtain a tissue preservation solution, and then mix the tissue preservation solution with PBS evenly according to the ratio shown in Table 1 to obtain the aborted tissue preservation solution.

[0067] Table 1 Preparation of the aborted tissue preservation solution

[0068]

[0069] Example 2: Preservation of the effluent tissue under different conditions

[0070] 1. Select 3 cases of effluent tissue from the sample library (the first extraction was QC and the DNA integrity was good). Cut out a part from each case and divide the excised part into 12 equal parts (the size of a soybean). Take 2 parts from each and store them at -25°C to -15°C (as the control group).

[0071] 2. Take 5 ml of the effluent tissue preservation solution prepared in Example 1, mix it well, and then aliquot 800 μl into 1.5 ml (or 2 ml) EP tubes.

[0072] 3. Distribute the remaining 30 samples in Step 1 into the corresponding preservation solution EP tubes in Step 2 according to Table 2. Invert and mix well to completely immerse the effluent tissue in the effluent preservation solution, and store it at room temperature of 25°C. Each case of effluent has 10 treatment methods. As shown in Table 2, extract the samples after storing them at room temperature for 3 days or 5 days, and simultaneously extract the control group stored at -25°C to -15°C (the extraction conditions are kept consistent).

[0073] 4. QC: Perform quality control on the integrity (the loading amount is kept consistent), concentration, and purity of the DNA. According to the QC results, select the optimal effluent tissue preservation solution formula (preservation effect, cost, etc.), and scale up production (sterile).

[0074] 5. According to the effluent collection kit provided by the market, use 25 ml centrifuge tubes (sterile consumables, or sterilized), add 12.5 ± 2.5 ml of the tested effluent tissue preservation solution to each tube (sterile operation), and package and deliver to the material warehouse.

[0075] Table 2 Preservation of the effluent tissue under different conditions

[0076]

[0077]

[0078] Compared with the control group (serial numbers 1 - 3), when stored for 3 days (serial numbers 4 - 18), Figure 1 The results show that the samples with serial numbers 10 - 18 did not degrade, and the sample tissue had good stability. However, because the tissue preservation solution was expensive, Mix1 (PBS: tissue preservation solution = 9:1) was the optimal option, which could greatly reduce the cost of the effluent tissue preservation solution on the premise of maintaining the stability of the effluent tissue.

[0079] Compared with the reference (serial numbers 19 - 21), when stored for 5 days (serial numbers 22 - 36), Figure 2The results show that Mix 1 with serial numbers 29 - 30 and the pure tissue preservation solution with serial numbers 35 - 36 have less degradation. Similarly, because the tissue preservation solution is relatively expensive, Mix 1 (PBS: tissue preservation solution = 9:1) is the optimal option, which can greatly reduce the cost of the tissue preservation solution for the aborted tissue under the premise of maintaining the stability of the aborted tissue.

[0080] When the preservation time is 3 days, the degradation of the Mix 1 samples is the same as that of the control group under the frozen state, and no degradation occurs. When the preservation time is 5 days, only 1 case of the Mix 1 samples has slight degradation, which is basically the same as that of the control group under the frozen conditions. Thus, it can be seen that the tissue preservation solution for the aborted tissue of the present invention can stably preserve the aborted tissue at room temperature, which is convenient for long-distance transportation.

[0081] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can also be made on the basis of the above embodiments. Similarly, the technical features of the above embodiments can also be arbitrarily combined to form other embodiments of the present invention that may not be clearly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. A tissue preservation solution for aborted tissues, characterized in that, The aborted tissue preservation solution is a mixed solution of PBS and tissue preservation solution, and the ratio of PBS to tissue preservation solution is 15:1 - 1:1; The tissue preservation solution is a mixed solution of DMSO and EDTA, and the ratio of DMSO to EDTA is 1:1 - 1:

10.

2. The tissue preservation solution for aborted tissues according to claim 1, wherein The ratio of PBS to tissue preservation solution is 9:

1.

3. The tissue preservation solution for aborted tissues according to claim 1, wherein The ratio of DMSO to EDTA is 1:

4.

4. A method for preparing a tissue preservation solution for aborted products according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: (1) Mix DMSO and EDTA evenly to obtain the tissue preservation solution; (2) Mix PBS and the tissue preservation solution obtained in step (1) evenly to obtain the aborted tissue preservation solution.

5. The preparation method according to claim 4, wherein: In step (1), the ratio of DMSO to EDTA is 1:1 - 1:10; In step (2), the ratio of PBS to tissue preservation solution is 15:1 - 1:

1.

6. The preparation method according to claim 5, wherein: In step (1), the ratio of DMSO to EDTA is 1:4; In step (2), the ratio of PBS to tissue preservation solution is 9:

1.

7. An application of the aborted tissue preservation solution according to any one of claims 1 - 3 or the aborted tissue preservation solution prepared by the preparation method according to any one of claims 4 - 6 in the preservation and transportation of aborted tissue.

8. The application according to claim 7, wherein The preservation is to completely immerse the aborted tissue after being taken out of the body in the aborted tissue preservation solution and preserve it at room temperature.

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