CN116616277B - Menstrual blood preservation solution and preparation method and application thereof - Google Patents
Menstrual blood preservation solution and preparation method and application thereof Download PDFInfo
- Publication number
- CN116616277B CN116616277B CN202310898602.2A CN202310898602A CN116616277B CN 116616277 B CN116616277 B CN 116616277B CN 202310898602 A CN202310898602 A CN 202310898602A CN 116616277 B CN116616277 B CN 116616277B
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- China
- Prior art keywords
- menstrual blood
- stem cells
- preservation solution
- sodium citrate
- ethylenediamine tetraacetic
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- 239000003761 preservation solution Substances 0.000 title claims abstract description 51
- 238000004555 blood preservation Methods 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title abstract description 10
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 27
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims abstract description 27
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The application discloses a menstrual blood preservation solution and a preparation method and application thereof, belonging to the field of biological agents. The menstrual blood preservation solution comprises ethylenediamine tetraacetic acid, anhydrous sodium citrate, an anti-adsorption agent, a preservative, pyruvate, magnesium salt and 0.9% physiological saline. The combination of ethylenediamine tetraacetic acid and sodium citrate dehydrate can deactivate calcium ions that cause coagulation of the menstrual blood, thereby preventing coagulation of the menstrual blood due to the presence of calcium ions. In addition, the ethylenediamine tetraacetic acid and the dehydrated sodium citrate have no cytotoxicity, and the combination of the ethylenediamine tetraacetic acid and the dehydrated sodium citrate can reduce the aggregation or degradation of the endometrium stem cells, and the morphology and the activity of the endometrium stem cells are not influenced. The anti-adsorption agent is used for preventing the endometrium stem cells from adhering to the wall surface of the container, and the preservative can prevent the endometrium stem cells from cracking and reducing the activity during the preservation period. The menstrual blood preservation solution of the present application is used for preserving menstrual blood, and after 14 days, endometrium stem cells in menstrual blood can maintain more than 95% of activity.
Description
Technical Field
The application relates to the field of biological agents, in particular to a menstrual blood preservation solution and a preparation method and application thereof.
Background
Adult stem cells are present in individual tissue organs, and they are capable of maintaining homeostasis, repairing organ damage due to disease. One of the commonly used multipotent adult stem cells is bone marrow mesenchymal stem cells, however, it has a certain invasive nature in the isolation process and is easy to cause discomfort to the patient, and umbilical cord blood stem cells are also an important source of adult stem cells, but have defects in source and acquisition opportunity due to the dependence of the maternal pregnancy. Other adult stem cells, such as adipose stem cells, amniotic fluid stem cells, umbilical cord stem cells, are not yet fully viable and have limited regenerative capacity, which limits their use to some extent, and thus the search for a non-invasive and safe and reliable source of stem cells is currently a major challenge.
The highly proliferated stem cells derived from menstrual blood, namely menstrual blood-derived stromal stem cells, become a focus of attention, and menstrual blood discharged by women each month contains a large amount of stem cells (endometrial stem cells), has an expansion capacity 30 times that of autologous bone marrow stem cells, and has very remarkable effects in nerve repair, tumor inhibition, beauty, aging resistance and the like. Because of the special relatedness of females in the home (orthotopic), their high histocompatibility and antigenicity are halved, so that stem cells from females themselves can benefit multiple family members. Although menstrual blood is collected in many times, how to store and transport the stem cells collected by discharging the menstrual blood in many times maintains the collected menstrual blood in a low temperature state so as to avoid the decrease of cell activity. Is a subject of constant study by those skilled in the art, but reports on this are not yet known. At present, although the preparation and storage methods of mesenchymal stem cells derived from menstrual blood are carried out by separating the mesenchymal stem cells from menstrual blood, purifying and culturing the mesenchymal stem cells, and then freezing the mesenchymal stem cells, the activity of the mesenchymal stem cells is reduced during the period, and even if the mesenchymal stem cells are stored, the activity of the mesenchymal stem cells cannot be maintained.
In summary, in the menstrual blood preservation solution adopted in the prior art, the cell morphology of the endometrial stem cells is easy to change and crack with the lapse of time in the storage process, the activity is reduced, the effect is general even if the menstrual blood preservation solution is preserved, the conventional menstrual blood preservation solution generally needs to be added with an anticoagulant to prevent the coagulation of menstrual blood during the storage process, and the anticoagulant contains cytotoxicity, so that the activity of the endometrial stem cells is easy to be influenced.
Disclosure of Invention
The application mainly aims to provide a menstrual blood preservation solution, a preparation method and application thereof, and solves the technical problems that an anticoagulant used by the conventional menstrual blood preservation solution has cytotoxicity, is easy to influence the activity of endometrium stem cells, and the morphology of the endometrium stem cells is easy to deform and break during preservation to influence the activity.
In order to achieve the above object, the present application provides a menstrual blood preservation solution comprising the following components: ethylenediamine tetraacetic acid, anhydrous sodium citrate, anti-adsorbent, preservative, pyruvate, magnesium salt and 0.9% physiological saline.
In some embodiments of the application, the anti-adsorption agent comprises at least one of polyvinyl alcohol, trehalose.
In some embodiments of the application, the preservative comprises at least one of imidazolidinyl urea, diazolidinyl urea, bisimidazolidinyl urea, sodium hydroxymethyl glycine, dimethylol urea, sodium hydroxymethyl glycine.
In some embodiments of the application, the magnesium salt comprises magnesium chloride.
In some embodiments of the present application, the ethylenediamine tetraacetic acid, anhydrous sodium citrate, anti-adsorption agent, preservative, pyruvate and 0.9% physiological saline are added in the following amounts based on the total volume L of 0.9% physiological saline:
ethylenediamine tetraacetic acid: 20g/L to 30g/L;
anhydrous sodium citrate: 100g/L to 150g/L;
anti-adsorption agent: 20g/L to 50g/L;
preservative: 1g/L to 10g/L;
pyruvate: 30g/L to 50g/L;
magnesium salt: 10g/L to 50g/L.
In some embodiments of the application, the menstrual fluid further comprises at least one of amino acids and vitamins.
In some embodiments of the application, the amino acid is added in an amount of 50g/L to 100g/L based on the total volume L of the physiological saline of 0.9%.
In some embodiments of the application, the vitamin is added in an amount of 50g/L to 100g/L based on the total volume L of the physiological saline of 0.9 percent.
In order to achieve the above object, the present application also provides a method for preparing a menstrual blood preservation solution, the method comprising the steps of: dissolving ethylenediamine tetraacetic acid, anhydrous sodium citrate, an anti-adsorbent, a preservative, pyruvate and magnesium salt into 0.9% physiological saline, uniformly stirring, and sterilizing to obtain the menstrual blood preservation solution.
In some embodiments of the present application, at least one of an amino acid and a vitamin is further added in the step of dissolving ethylenediamine tetraacetic acid, anhydrous sodium citrate, an anti-adsorbent, a preservative, and pyruvate into 0.9% physiological saline.
In order to achieve the above object, the present application also provides an application of the above menstrual blood preservation solution in the menstrual blood preservation field.
The application has the beneficial effects that:
according to the application, the calcium ions which cause the coagulation of the menstrual blood can be deactivated by the cooperation of the ethylenediamine tetraacetic acid and the anhydrous sodium citrate, so that the coagulation of the menstrual blood caused by the existence of the calcium ions can be prevented, the aggregation or degradation of the endometrium stem cells can be prevented, the deformation of the morphology of the endometrium stem cells caused by the aggregation or the inactivation caused by the degradation can be avoided, in addition, the ethylenediamine tetraacetic acid and the anhydrous sodium citrate have no cytotoxicity, and the activity of the endometrium stem cells is not influenced.
The anti-adsorption agent is used for preventing the endometrium stem cells from adhering to the wall surface of the container, inhibiting the growth of bacteria and fungi by adding the preservative, preventing the menstrual blood from putrefaction, preventing the endometrium stem cells from cracking during the preservation period, keeping the endometrium stem cells in good form and ensuring good biological activity for a long time.
The magnesium salt in the menstrual blood preservation solution can improve and maintain the activity of plasmin, strengthen the dissolution of fibrin and prevent the coagulation of menstrual blood.
When the menstrual blood is stored in the menstrual blood preservation solution, the endometrium stem cells can be in a suspension state in the menstrual blood preservation solution, and can not adhere to the wall surface of the container, and can not be aggregated due to the coagulation of the menstrual blood or the conventional sedimentation of the cells, so that the endometrium stem cells can keep better cell morphology and bioactivity.
Drawings
For a clearer description of embodiments of the application or of solutions in the prior art, the following brief description of the drawings is given for the purpose of illustrating the embodiments or the solutions in the prior art, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained from the structures shown in these drawings without the need for inventive effort for a person skilled in the art.
Fig. 1 is a schematic diagram of a process for preparing a menses preservation solution according to the present application.
The achievement of the objects, functional features and advantages of the present application will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The description as it relates to "first", "second", etc. in the present application is for descriptive purposes only and is not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be considered to be absent and not within the scope of protection claimed in the present application.
The application provides a menstrual blood preservation solution, which comprises the following components:
ethylenediamine tetraacetic acid, anhydrous sodium citrate, an anti-adsorbent agent, a preservative, sodium pyruvate, magnesium salt and 0.9% physiological saline.
About half of the menstrual blood is blood which contains sodium, calcium, phosphate, iron and chloride, and in addition, cervical mucus, vaginal secretions, endometrial tissue, dielectrics, and a variety of proteins including glycoproteins. Furthermore, prostaglandins and a large amount of plasmin derived from endometrium are contained in menstrual blood, and plasmin has an effect of dissolving fibrin, so that coagulation of menstrual blood does not occur, but plasmin is highly likely to be destroyed and deactivated during extraction of menstrual blood or during storage of menstrual blood, and it is difficult to continue to exert an effect of keeping menstrual blood from coagulating. At the same time, calcium ions in menstrual blood also tend to cause coagulation of menstrual blood.
The ethylenediamine tetraacetic acid and the anhydrous sodium citrate have no cytotoxicity, do not influence the activity of the endometrium stem cells, and can inactivate calcium ions which are easy to cause the coagulation of menstrual blood when being used in a compound way, thereby preventing the coagulation of menstrual blood. In addition, the ethylene diamine tetraacetic acid and the anhydrous sodium citrate can be used together to prevent the aggregation or degradation of the endometrium stem cells, and can avoid the deformation and cracking of the morphology of the endometrium stem cells caused by aggregation or the inactivation caused by degradation.
In some embodiments, the amount of ethylenediamine tetraacetic acid added is 20g/L to 30g/L, and the amount of anhydrous sodium citrate is 100 to 150g/L, based on the total volume L of 0.9% physiological saline, e.g., any one of 20g/L to 30g/L, such as 20g/L, 22g/L, 23g/L, 25g/L, 26g/L, 27g/L, 28g/L, 29g/L, 30g/L, etc.; the amount of sodium citrate anhydrous may be any of 100g/L, 110g/L, 115g/L, 120g/L, 125g/L, 130g/L, 135g/L, 140g/L, 145g/L, etc. within the range of 100g/L to 150 g/L. In the above-mentioned addition amount range, the menstrual blood preservation solution can deactivate calcium ions in menstrual blood, and is not easy to cause the decrease of activity caused by aggregation, degradation and deformation of endometrium stem cells.
In the application, the anti-adsorption agent can play a role in preventing the endometrium stem cells from being adsorbed on the container wall.
In some embodiments, the anti-adsorption agent comprises at least one of polyvinyl alcohol, trehalose. The adsorbents can prevent the endometrium stem cells from being adsorbed on the wall surface of the container, have no cytotoxicity and are not easy to destroy the biological activity of the endometrium stem cells.
In some embodiments, the amount of the anti-adsorption agent added is 20g/L to 50g/L based on the total volume L of 0.9% physiological saline, for example, the amount may be any one of the ranges of 20g/L to 50g/L, such as 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, etc.
In the present application, the preservative can prevent rupture of endometrium stem cells, affecting the biological activity thereof. In some embodiments, the preservative comprises imidazolidinyl urea, diazoimidazolidinyl urea, bisimidazolidinyl urea, sodium hydroxymethyl glycine, dimethylol urea, sodium hydroxymethyl glycine, which can inhibit the growth of bacteria and fungi, prevent the putrefaction of menstrual blood, prevent the rupture of endometrium stem cells during the preservation period, keep good morphology and ensure better bioactivity.
In some embodiments, the preservative is added in an amount of 1g/L to 10g/L based on the total volume L of 0.9% physiological saline, for example, the preservative may be added in an amount of any one of 1g/L to 10g/L, such as 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, etc. Under the condition of the addition amount, the preservative can protect the endometrium stem cells from rupture to influence the activity, and can inhibit the growth of fungi and bacteria in menstrual blood, so that the endometrium stem cells can maintain better biological activity under the long-time preservation of the menstrual blood.
In the application, pyruvate can slow down metabolism of endometrium stem cells and maintain biological activity for a long time.
In some embodiments, the amount of pyruvate added is 30g/L to 50g/L based on the total volume L of 0.9% physiological saline, for example, the amount of pyruvate added may be any one of the ranges of 30g/L to 50g/L, such as 30g/L, 32g/L, 35g/L, 40g/L, 45g/L, 48g/L, 50g/L, etc. In the addition amount range, the effect of the pyruvate in slowing down the metabolism of the endometrium stem cells can be well exerted, and the biological activity of the endometrium stem cells can be maintained for a long time.
In the application, the magnesium salt can improve and maintain the activity of plasmin, strengthen the dissolution of fibrin and prevent coagulation of menstrual blood. In some embodiments, the magnesium salt comprises magnesium chloride, wherein magnesium ions in the magnesium chloride can increase and maintain plasmin activity, enhance fibrinolysis, and prevent coagulation of menstrual blood.
The application is not limited to the amount of magnesium salt added, and in some embodiments, the amount of magnesium salt added is in the range of 10g/L to 50g/L based on the total volume L of 0.9% physiological saline, for example, any one of 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L and the like is added, and in the above addition amount range, magnesium salt can be dissolved in the menses preservation solution to provide a large amount of magnesium ions, improve and maintain the activity of plasmin, strengthen the dissolution of fibrin and prevent menses from coagulation.
Under the combined action of the ethylenediamine tetraacetic acid, the anhydrous sodium citrate, the anti-adsorbent, the preservative, the pyruvate and the magnesium salt, the endometrium stem cells can be in a suspension state in the menstrual blood preservation solution, can not adhere to the wall surface of the container, can not be aggregated due to the coagulation of the menstrual blood or the conventional sedimentation of the cells, and can keep better cell morphology and bioactivity.
In addition, at least one of amino acid and vitamin can be added into the menstrual blood preservation solution, the amino acid and the vitamin can provide nutrition for the endometrium stem cells, and the metabolism speed of the endometrium stem cells is slowed down by cooperating with pyruvate, so that the preservation period of menstrual blood is prolonged.
In some embodiments, the amino acid is added in an amount of 50g/L to 100g/L based on the total volume L of the menses preservation solution.
In some embodiments, the vitamin is added in an amount of 50g/L to 100g/L based on the total volume L of the physiological saline of 0.9%.
In some embodiments, the menstrual blood preservation solution contains amino acids and vitamins, and the combination of the amino acids and the vitamins can provide better nutrients for the endometrium stem cells, slow down the metabolism speed of the endometrium stem cells and improve the preservation period of menstrual blood.
In some embodiments, the menstrual blood preservation solution of the present application may further contain a Chinese medicine extract, wherein the Chinese medicine extract includes at least one of Ligusticum wallichii, pseudo-ginseng and myrrh. The Chinese medicinal extract is beneficial to improving and maintaining the activity of plasmin, promoting fibrinolysis and preventing coagulation of menstrual blood.
In some embodiments, the Chinese medicinal extract includes a Chinese medicinal water extract, which may be understood as a solid obtained by dehydrating and drying an extract using an aqueous solution as a solvent. The traditional Chinese medicine water extract is only obtained by extracting with water solution, does not use other organic solvents, and is not easy to cause damage to endometrium stem cells.
In some embodiments, the extraction method of the above-mentioned Chinese medicine water extract is as follows:
pulverizing the above materials, adding 10-20 times of water, mixing, decocting, filtering, concentrating, and drying to obtain fine granule water extract.
The above Chinese medicinal water extract can be rapidly dissolved to obtain uniform menstrual blood preservation solution.
In some embodiments, the amount of the extract of the Chinese medicine added is 10g/L to 15g/L based on the total volume L of the physiological saline of 0.9%, for example, any one of 10g/L to 15g/L such as 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, etc.
The traditional Chinese medicine extract can sometimes precipitate in the menstrual blood preservation solution, and in some embodiments, the menstrual blood preservation solution can also contain a nonionic surfactant, so that the nonionic surfactant has a good compatibilization effect, the precipitation of the traditional Chinese medicine extract is reduced, the solubility of the nonionic surfactant is good, the nonionic surfactant is not easily influenced by acid and alkali, and the nonionic surfactant can be mixed with other components in the menstrual blood preservation solution for use, and has good compatibility.
In some embodiments, the nonionic surfactant comprises a fatty acid sucrose polyester, the fatty acid sucrose polyester has no cytotoxicity, is not easy to destroy the activity of endometrium stem cells, has a better compatibilizing effect, can slow down the problem of precipitation of menstrual blood preservation liquid, is beneficial to exerting the beneficial effect of activating plasmin by the traditional Chinese medicine extract, and can also prevent the menstrual blood preservation liquid from easily precipitating to cause a large number of endometrium stem cells to be deposited at the bottom of a container to destroy the cell structure of the endometrium stem cells and influence the biological activity.
In some embodiments, the nonionic surface is added in an amount of 3g/L to 10g/L, based on the total volume L of 0.9% physiological saline, which may be 3g/L, 5g/L, 8g/L, 10g/L, etc.
In some embodiments, the volume ratio of the menstrual fluid preservation solution and menstrual fluid of the present application may be 10: (1-3), e.g., 10:1, 10:1.5, 10:2, 10:2.5, 10:3. The menstrual blood may be an extract of endometrial stem cells obtained by separating and purifying menstrual blood, or may be menstrual blood stock solution.
In addition, the application also provides a preparation method of the menstrual blood preservation solution, referring to fig. 1, comprising the following steps:
step S10, dissolving ethylenediamine tetraacetic acid, anhydrous sodium citrate, an anti-adsorption agent, a preservative, pyruvate and magnesium salt into 0.9% physiological saline, and uniformly stirring;
step S20, sterilizing to obtain the menstrual blood preservation solution.
In some embodiments, the menstrual fluid of the present application is preserved at 4 ℃.
The application is not limited to the particular manner in which sterilization may be accomplished under conditions of high pressure and high temperature in some embodiments.
In some embodiments, the stirring speed may be 400-500rpm/min, and under the stirring condition, the components of the ethylenediamine tetraacetic acid, the dehydrated sodium citrate, the anti-adsorbent, the preservative and the pyruvate may be dispersed more uniformly.
In some embodiments, amino acids and/or vitamins may be added in step S10, which may provide nutrients to the endometrial stem cells, so that they may also maintain a good biological activity over a long period of storage.
In some embodiments, a herbal extract and a nonionic surfactant may also be added in step S10.
The technical scheme of the present application will be further described in detail with reference to the following specific examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure.
Examples 1 to 5
The preparation method of the menstrual blood preservation solutions of examples 1 to 5 is as follows:
the raw materials were weighed according to Table 1, dissolved in 1L of 0.9% physiological saline, stirred uniformly at a rotation speed of 400-500rpm, sterilized at high temperature under high pressure, and stored at 4 ℃.
The amount of the raw materials in Table 1 was based on 1L of 0.9% physiological saline.
Comparative example 1
Comparative example 1 a menstrual blood preservation solution was prepared by referring to the preparation method of example 1, except that ethylenediamine tetraacetic acid and anhydrous sodium citrate were not added, as specifically shown in table 1.
Comparative example 2
Comparative example 1 a menstrual blood preservation solution was prepared by the method of preparation of example 1, except that pyruvate was not added.
Comparative example 3
The menstrual blood preservation solution containing heparin sodium, and the menstrual blood preservation solution of comparative example 3 is phosphate buffer solution containing 2X penicillin, 2X streptomycin, 30ug/ml kanamycin, 40ug/ml sodium citrate, and 100U/ml heparin sodium.
TABLE 1 raw material compositions (g/L) of examples 1-5 and comparative examples 1-2 menstrual blood conserving solutions
Performance testing
1. The extracted menstrual blood was added to the menstrual blood preservation solutions obtained in examples 1 to 5 and comparative examples 1 to 3, respectively, at a weight ratio of 1:10.
2. The surface markers of the mesenchymal stem cells of the endometrium in the menstrual blood preservation solutions of examples 1-5 and comparative examples 1-3 are detected by using a flow cytometer, and the positive expression of the markers is that the surface markers of the mesenchymal stem cells are marked as CD29, CD44, CD73, CD90, CD105 and HLA-ABC. Positive marker expression data were measured on days 1, 3, 7, 10, and 14, respectively, and the results of the tests are shown in tables 2 to 6. Mesenchymal stem cell identification criteria promulgated by the international stem cell society ISCT: positive expression: CD29, CD44, CD73, CD90, CD105 not less than 95.0%; negative expression HLA-DR is less than or equal to 2.0 percent.
TABLE 2 day 1 flow cytometer detection results
TABLE 3 day 3 flow cytometer detection results
Table 4 day 7 flow cytometer detection results
Table 5 day 10 flow cytometer detection results
Table 6 day 14 flow cytometer detection results
As shown in tables 1 to 6, the menstrual blood preservation solutions prepared in examples 1 to 5 can maintain the activity of endometrium stem cells at least 95% after 14 days.
In comparative example 1, ethylenediamine tetraacetic acid and anhydrous sodium citrate were not added, and the coagulation phenomenon was easily caused in menstrual blood, and the rupture was easily caused by aggregation of endometrium stem cells, and thus the activity of endometrium stem cells was also affected, and after 14 days of preservation in the menstrual blood preservation solution of comparative example 1, the activity of endometrium stem cells was slightly decreased compared with that of example 1.
Comparative example 2 was free of pyruvate, and the activity of endometrium stem cells was also decreased after 14 days of storage.
In contrast, comparative example 3 the existing menstrual blood preservation solution containing heparin sodium, heparin sodium has cytotoxicity, and after 14 days of preservation, the activity of the endometrial stem cells is poorer than that of the endometrial stem cells after 14 days of preservation in example 1 of the application.
The foregoing description is only of the preferred embodiments of the present application, and is not intended to limit the scope of the application, but rather is intended to cover any equivalents of the structures or equivalent processes disclosed herein or in the alternative, which may be employed directly or indirectly in other related arts.
Claims (9)
1. A menstrual blood preservation solution, characterized in that the menstrual blood preservation solution comprises the following components: ethylenediamine tetraacetic acid, anhydrous sodium citrate, anti-adsorbent, antiseptic, pyruvate, magnesium salt, 0.9% physiological saline, chinese medicinal extract, and nonionic surfactant;
the Chinese medicinal extract comprises at least one of rhizoma Ligustici Chuanxiong, notoginseng radix, and Myrrha;
the nonionic surfactant is fatty acid sucrose polyester;
based on the total volume L of the 0.9% physiological saline, the additive amounts of the ethylenediamine tetraacetic acid, the anhydrous sodium citrate, the anti-adsorption agent, the preservative, the pyruvate, the magnesium salt, the traditional Chinese medicine extract and the nonionic surfactant are as follows:
ethylenediamine tetraacetic acid: 20g/L to 30g/L;
anhydrous sodium citrate: 100g/L to 150g/L;
anti-adsorption agent: 20g/L to 50g/L;
preservative: 1g/L to 10g/L;
pyruvate: 30g/L to 50g/L;
magnesium salt: 10g/L to 50g/L;
extract of traditional Chinese medicine: 10g/L to 15g/L;
nonionic surfactant: 3g/L to 10g/L.
2. The menstrual fluid according to claim 1, wherein the anti-adsorption agent comprises at least one of polyvinyl alcohol and trehalose.
3. The menstrual fluid according to claim 1, wherein the preservative comprises at least one of imidazolidinyl urea, diazolidinyl urea, bisimidazolidinyl urea, sodium hydroxymethyl glycine, dimethylol urea, sodium hydroxymethyl glycine.
4. The menstrual fluid according to claim 1, wherein the magnesium salt comprises magnesium chloride.
5. The menses preservation solution of claim 1, further comprising at least one of an amino acid and a vitamin.
6. The menstrual blood preservation solution according to claim 5, wherein the amino acid is added in an amount of 50g/L to 100g/L based on the total volume L of 0.9% physiological saline;
and/or, the addition amount of the vitamin is 50g/L-100g/L based on the total volume L of the physiological saline of 0.9 percent.
7. A method for preparing a menstrual blood preservation solution, which is characterized by comprising the following steps: dissolving ethylenediamine tetraacetic acid, anhydrous sodium citrate, an anti-adsorbent, a preservative, pyruvate, magnesium salt, a traditional Chinese medicine extract and a nonionic surfactant into 0.9% physiological saline, uniformly stirring, and sterilizing to obtain the menstrual blood preservation solution; the Chinese medicinal extract comprises at least one of rhizoma Ligustici Chuanxiong, notoginseng radix, and Myrrha;
the nonionic surfactant is fatty acid sucrose polyester;
based on the total volume L of the 0.9% physiological saline, the additive amounts of the ethylenediamine tetraacetic acid, the anhydrous sodium citrate, the anti-adsorption agent, the preservative, the pyruvate, the magnesium salt, the traditional Chinese medicine extract and the nonionic surfactant are as follows:
ethylenediamine tetraacetic acid: 20g/L to 30g/L;
anhydrous sodium citrate: 100g/L to 150g/L;
anti-adsorption agent: 20g/L to 50g/L;
preservative: 1g/L to 10g/L;
pyruvate: 30g/L to 50g/L;
magnesium salt: 10g/L to 50g/L;
extract of traditional Chinese medicine: 10g/L to 15g/L;
nonionic surfactant: 3g/L to 10g/L.
8. The method according to claim 7, wherein at least one of an amino acid and a vitamin is further added in the step of dissolving ethylenediamine tetraacetic acid, anhydrous sodium citrate, an anti-adsorption agent, a preservative, pyruvate, magnesium salt, and a Chinese medicinal extract in 0.9% physiological saline.
9. Use of the menstrual blood preservation solution according to any one of claims 1 to 6 in the menstrual blood preservation field.
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| CN113265443A (en) * | 2021-05-21 | 2021-08-17 | 深圳逗点医疗科技有限公司 | Blood free DNA protection reagent, protection method and blood collection tube |
| CN115381445A (en) * | 2021-05-24 | 2022-11-25 | 上海长一干细胞研究中心有限公司 | Menstrual blood collector for human menstrual blood, preservation solution and matched preservation and transportation device |
| CN115644170A (en) * | 2022-11-21 | 2023-01-31 | 北京赛普九洲科技发展有限公司 | Menstrual blood preserving fluid and preparation method and application thereof |
| CN116034987A (en) * | 2022-12-26 | 2023-05-02 | 首都医科大学 | Tissue and cell preservation solution, cell separation method and application thereof |
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| CN110612978A (en) * | 2019-09-26 | 2019-12-27 | 广东华夏健康生命科学有限公司 | Preservation solution for human menstrual blood and preparation method thereof |
| CN113265443A (en) * | 2021-05-21 | 2021-08-17 | 深圳逗点医疗科技有限公司 | Blood free DNA protection reagent, protection method and blood collection tube |
| CN115381445A (en) * | 2021-05-24 | 2022-11-25 | 上海长一干细胞研究中心有限公司 | Menstrual blood collector for human menstrual blood, preservation solution and matched preservation and transportation device |
| CN115644170A (en) * | 2022-11-21 | 2023-01-31 | 北京赛普九洲科技发展有限公司 | Menstrual blood preserving fluid and preparation method and application thereof |
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