CN110622954A - Preservation solution for human menstrual blood and preparation method thereof - Google Patents
Preservation solution for human menstrual blood and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Abstract
The invention belongs to the technical field of stem cell preservation, and provides a preservation solution for human menstrual blood and a preparation method thereof. The components of the preservation solution comprise normal saline, hirudin, glucose, hydroxyethyl starch, levofloxacin, multivitamin, glutathione and necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor, and the preparation method comprises the following steps: 1) preparing a solution A: adding glucose, hirudin and levofloxacin into physiological saline respectively; 2) preparing a solution B: respectively adding hydroxyethyl starch, vitamin complex and glutathione into physiological saline; 3) preparing a solution C: the liquid A and the liquid B are mixed, and then added with necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor, and the mixture is filtered by a filter membrane and stored at 4 ℃ for later use.
Description
Technical Field
The invention belongs to the technical field of stem cell preservation, and particularly relates to human menstrual blood preservative fluid and a preparation method thereof.
Background
Menstruation is regulated by the interaction of reproductive hormones of the hypothalamus, pituitary gland and ovary, and in the menstrual period and the proliferation period of the menstrual cycle, the level of estradiol and progesterone in blood is very low, so that the negative feedback effect on the adenohypophysis and the hypothalamus is weakened or eliminated, the secretion of gonadotropin-releasing hormone by the hypothalamus is increased, and then follicle stimulating hormone and luteinizing hormone secreted by the adenohypophysis are increased, so that the follicle develops and the estrogen secretion is gradually increased. At this point, estrogen in turn stimulates the endometrium into the proliferative phase. Luteinizing hormone increases the secretion of progestogen, resulting in ovulation. During this period both estrogen and progestin levels are elevated. This produces an enhanced negative feedback inhibition of the hypothalamus and adenohypophysis, which in turn reduces the levels of ovulation stimulating hormone and luteinizing hormone, resulting in a deterioration of the corpus luteum and thus in a reduction of the estrogen and progestin levels. The endometrium is deprived of the support of these two hormones and is exfoliated and bleeded, i.e. menstruation occurs. At this point, the estrogen and progestin decrease begins the next menstrual cycle again.
The components of menses are mainly blood (3/4 arterial blood, 1/4 venous blood), fragments of endometrial tissue and various active enzymes and biological factors, wherein the fibrinolytic enzyme makes the menstrual blood liquid and non-coagulated, and the prostaglandin acts to contract the uterus. Menstrual blood rapidly coagulates in a short time under the condition of in vitro; the viability decreases, which directly affects the cell quality of endometrial stem cells in menstrual blood. The viability of endometrial stem cells is directly related to menstrual blood preservation; coagulation after the menstrual blood is isolated, influence of hormones in the menstrual blood and the like on the cell quality; how to avoid the above becomes the primary solution. In addition, these problems directly restrict success of in vitro culture of endometrial stem cells derived from menstrual blood; therefore, a menstrual blood preserving fluid needs to be developed, which can meet the anticoagulation and bacteriostasis effects on one hand, and can also meet the requirement that the endometrial stem cells with sufficient quantity and good quality can be extracted after the menstrual blood is preserved in vitro for 48 hours on the other hand.
In the prior art, substances such as penicillin, amino acid, human serum albumin and the like are generally added into the menstrual storage fluid for storing isolated tissues, but due to the reasons of single nutrient components, high cost, high antibiotic concentration and the like, although antibiotics can reduce the contamination probability, certain toxicity exists on placental stem cells, and the phenomena of low activity, even death and the like of the seed cells occur in long-term storage or transportation. Then, in order to further improve the composition system of the preservation solution, a plurality of amino acids and antibiotics are added on the basis of the buffer solution formed by inorganic ions to maintain the activity of isolated cells and reduce the infection rate. Although the activity of seed cells is well maintained for a long time by the measures, the components of the preservation solution are complex, the steps of weighing, sterilizing and the like are required for preparation, the operation process is complicated, and the portable application cannot be realized. Therefore, in order to meet the clinical application needs, it is urgent to develop a placenta preservation solution which contains multiple nutrients and is convenient to prepare.
Disclosure of Invention
In view of the above problems, an object of the present invention is to provide a preservation solution for human menstrual blood and a preparation method thereof, which can maintain the activity of endometrial stem cells from menstrual blood without causing functional variation of the cells, and in the prior art, albumin and the like are often used as a nutrition source for maintaining the activity of placental mesenchymal stem cells.
The technical content of the invention is as follows:
the invention provides a preserving fluid for human menstrual blood, which comprises the components of normal saline, hirudin, glucose, hydroxyethyl starch, levofloxacin, vitamin complex, glutathione and a necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor.
Furthermore, the concentration of the hirudin is (1-5) x hirudin, the hirudin is one of the most significant and most studied components extracted from leeches and salivary glands thereof, and is a small molecular protein (polypeptide) consisting of 65-66 amino acids, and the hirudin has extremely strong inhibition effect on thrombin and is the strongest natural specific thrombin inhibitor discovered so far;
the buffer solution based on the physiological saline is purchased from a reagent company, so that the stability of a storage solution buffer system can be ensured, and the quality difference caused by artificial configuration is avoided;
the glucose is used as a basic nutrient component of the endometrial stem cells, can be directly absorbed and utilized by the cells, improves the resistance of the cells, and maintains the activity of the cells;
the concentration of the hydroxyethyl starch is 1-5%, and the hydroxyethyl starch can maintain the osmotic pressure of a preservation solution, so that the endometrial stem cells are preserved in an isotonic and isopressure environment for the activity of the placental mesenchymal stem cells;
the concentration of the levofloxacin is (1-2) multiplied by levofloxacin, the levofloxacin quinolone drug is one of medicines, the levofloxacin quinolone drug has broad-spectrum antibacterial action and strong antibacterial action, can effectively inhibit various bacterial communities in the vagina of women, and has strong antibacterial activity on most enterobacteriaceae bacteria, such as escherichia coli, klebsiella, proteus, salmonella, shigella, haemophilus influenzae, legionella pneumophila, neisseria gonorrhoeae and other gram-negative bacteria; it also has antibacterial effect on gram-positive bacteria such as Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, etc., mycoplasma pneumoniae, and chlamydia pneumoniae;
the concentration of the necroptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor is 5-10 ng/mL, the necroptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor can directly and constantly perform apoptosis, preserve the biological characteristics of cells, comprehensively improve the survival rate of the cells and maintain the survival of the cells;
the concentration of the compound vitamin is 1-5 mg/mL;
the concentration of the glutathione is 1-5 mg/mL, the glutathione is a powerful antioxidant and exists in each cell of a body, redundant free radicals can be eliminated, and the antioxidation effect is achieved.
The invention also provides a preparation method of the human menstrual blood preservative fluid, which comprises the following steps:
1) preparing a solution A: adding glucose, hirudin and levofloxacin into physiological saline to obtain solution A;
2) preparing a solution B: respectively adding hydroxyethyl starch, vitamin complex and glutathione into physiological saline to obtain solution B, wherein the combined use of the vitamin complex and the glutathione can comprehensively improve the antioxidant function of cells;
3) preparing a solution C: mixing solution A and solution B, adding necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor to obtain solution C, filtering with filter membrane, and storing at 4 deg.C, and storing menstrual blood at low temperature to avoid nutritional component denaturation and maintain bioactivity of seed cells.
The volume ratio of the liquid A and the liquid B mixed in the step 3) is 1:1, and the pore size of the filter membrane is 0.22 mu m.
The invention has the following beneficial effects:
the preservation solution for human menstrual blood disclosed by the invention takes glucose as a basic nutrient component, takes physiological saline as a buffer solution, and also comprises different anti-apoptosis preparations, so that the stress resistance of cells can be enhanced, the activity of endometrial stem cells from menses can be well maintained under a proper preservation environment and nutrition supply, the long-time survival rate of in-vitro placenta in the preservation solution is ensured, the functional variation of the cells is not caused, and the in-vitro menses is anticoagulated and bacteriostatic, and the preservation solution disclosed by the invention can preserve in-vitro menstrual blood for 48 hours, can maintain the original biological characteristics of the endometrial stem cells within 48 hours, maintain the quality of the cells, solve the technical problem of in-vitro preservation of menstrual blood, and meanwhile, the separated cells are not differentiated, stable in cell quality and high in purity, and can be produced in a large scale.
The preserving fluid has simple preparation steps, can be prepared in large quantities in a short time, and has the advantages of low cost, stable components, safety and no toxic or side effect.
Drawings
FIG. 1 is a flow identification chart of a control group for extracting endometrial stem cells after the preservation solution of the invention is used for menstrual blood;
FIG. 2 is a flow identification chart of a sample group from which endometrial stem cells are extracted after a preservation solution of the present invention is applied to menstrual blood;
FIG. 3 is a diagram showing the cell morphology of endometrial stem cells extracted after the preservation solution of the invention is used for menstrual blood.
Detailed Description
The present invention is described in further detail in the following description of specific embodiments and the accompanying drawings, it is to be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and modifications thereof by those skilled in the art after reading this disclosure that are equivalent to the above described embodiments.
All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.
Examples
A preparation method of a preservative fluid for human menstrual blood comprises the following steps:
1) preparing a solution A: adding glucose, hirudin and levofloxacin into 0.9% normal saline to obtain solution A, respectively, to obtain 4% glucose, (2-10) x hirudin, and (2-4) x levofloxacin;
2) preparing a solution B: respectively adding hydroxyethyl starch, vitamin complex and glutathione into 0.9% normal saline to obtain solution B, and obtaining 2-10% hydroxyethyl starch, 2-10 mg/mL vitamin complex and 2-10 mg/mL glutathione;
3) preparing a solution C: mixing the solution A and the solution B according to a volume ratio of 1:1, adding Ac-DEVD-CHO to obtain a solution C, obtaining a preservation solution with a final concentration of 2% glucose, (1-5) x hirudin, (1-2) x levofloxacin, 1-5% hydroxyethyl starch, 1-5 mg/mL multivitamin, 1-5 mg/mL glutathione and 5-10 ng/mL necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor, filtering the preservation solution with a 0.22 mu m filter membrane, and preserving the preservation solution at an ambient temperature of 4 ℃ for later use.
Test example 1
1) Human menstrual blood collection
Taking menstrual blood of a woman of 18-36 years old, regular menstrual cycle and 5-7 menstrual period time as an experimental object, collecting menstrual blood within 12-36 hours by taking red as a time starting point for menstruation, wiping vulva with sterile gauze before collection, placing a menstrual cup into a vagina, taking out after 15-30 minutes, sucking the menstrual blood by using a sterile suction pipe, softly and fully mixing the menstrual blood with the preservation solution prepared in the embodiment according to the volume ratio of 1:10, and preserving at 4 ℃;
2) human menstrual blood preservation
The collected menstrual blood enters the separation culture of cells within 0h, 24h and 48h, and the preservation process is kept at 4 ℃;
3) culture of endometrial stem cells
According to the lymph separation fluid: menstrual blood volume ratio is 1: 1; adding the lymphocyte separating solution into each tube by using a disposable pipette, and slowly transferring the diluted blood to the surface of the lymphocyte separating solution by using the disposable pipette or a Pasteur pipette so as to form a clear interface between the lymphocyte separating solution and the lymphocyte separating solution. The tube was transferred to a centrifuge and centrifuged at 700g for 15 min. After centrifugation, the buffy coat was aspirated and transferred to another 50mL centrifuge tube, RPMI 1640 medium was added to 40mL using a disposable pipette, the PBMC was resuspended and centrifuged at 500g for 5 min. Discarding the supernatant, resuspending the cell pellet with high-sugar DMEM + 10% FBS, inoculating to a 10cm dish, transferring to 37 deg.C, and 5.0% CO2CO with a humidity of 95.0%2Culturing in an incubator.
4) Quality evaluation of endometrial stem cells
a. And (3) morphology detection: taking the endometrial stem cells in the logarithmic growth phase, and observing the morphology of the cells under an inverted microscope.
b. Proliferation rate: after 48 hours of cell inoculation, carrying out liquid changing treatment; taking a dish of cells every 48 hours for enzymolysis until the fusion degree of the batch of cells reaches 100%, and counting the number of the cells to obtain the cell proliferation speed.
c. And (3) flow type result detection: taking the endometrium stem cells in logarithmic growth phase, and adjusting the cell density to be 1 multiplied by 106cell suspension of cells, respectively taking monoclonal antibodies of anti-human CD105, CD90, CD73, CD45, CD34 and HLA-DR 2.5 muL, adding 500 muL of cell suspension, incubating at room temperature for 20min in a dark place to serve as a sample group, simultaneously setting up an isotype control as a control group, centrifuging at 1500r/min for 5min, discarding supernatant, washing with 10% FBS-containing PBS for 2 times, resuspending with 500 muL 1640, and detecting on a machine (the sample group contains fluorescent antibody, the isotype control is set to beAs a limit to determine the specific purity of the cell);
fig. 1 is a flow identification diagram of a control group, fig. 2 is a flow identification diagram of a sample group, and from the flow result, the endometrial stem cells highly express CD73, CD90 and CD105, and the expression rates are 99.9%, 99.9% and 97.2%, respectively; low expression of HLA-DR, CD45, CD19, CD11b and CD34 at 0.1%, 0.0%, 0.1%, 0.4% and 0.2%, respectively. The surface antigen characteristics of the mesenchymal stem cells (the identification standard of the mesenchymal stem cells issued by the International Stem cell society ISCT, namely positive expression of CD73, CD90 and CD105 is more than 95.0 percent, and negative expression of HLA-DR, CD45, CD11b, CD34 and CD19 is less than 2.0 percent) are met, which indicates that the endometrial stem cells do not have the phenomenon of differentiation and still maintain the characteristics of the stem cells.
Test example 2
The menstrual blood was stored in the storage solution prepared in example 1 according to test example 2, the extracted endometrial stem cells were subjected to amplification culture, and then 3 samples (sample 1, sample 2, and sample 3) were subjected to cell viability and morphology detection, as shown in the following table (table 1), showing that the storage solution of this example has a significant effect on cell storage.
TABLE 1 measurement of cell viability of samples
| Group of | Rate of activity |
| Endometrial stem cell sample 1 | 94.07％ |
| Endometrial stem cell sample 2 | 96.55％ |
| Endometrial stem cell sample 3 | 95.73％ |
Fig. 3 is a diagram of morphological detection of cells, which shows that the endometrial stem cells are resistant to staining, dead cells are stained blue, and viable cells are resistant to staining under trypan blue detection, and the endometrial stem cells still keep vitality under the action of the preservation solution of the embodiment.
Test example 3
4 groups of different preservation solutions are prepared for storing menstrual blood, the survival rates of mononuclear cells in the menstrual blood under different preservation times are detected and are shown in table 2, and the four groups of preservation solutions are respectively a blank control group, a preservation solution A, a preservation solution B and a preservation solution C, and the components of the four groups of preservation solutions are respectively as follows:
blank control group: 0.9% physiological saline, 2 × levofloxacin;
preservation solution a: 0.9% of physiological saline contains 1 x hirudin, 2% of glucose, 2% of hydroxyethyl starch and 2 x levofloxacin;
preservation solution B: 0.9% physiological saline contains 1 × hirudin, 2% glucose, 2% hydroxyethyl starch, 2 × levofloxacin, 5mg/mL multivitamin, and 5mg/mL glutathione;
preservation solution C: 0.9% physiological saline contains 1 × hirudin, 2% glucose, 2% hydroxyethyl starch, 2 × levofloxacin, 5mg/mL multivitamin, 5mg/mL glutathione, and 10ng/mL necroptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor.
TABLE 2 survival rates of mononuclear cells in menstrual blood at different storage times
Indicates significant difference compared to control, P < 0.05;
indicates a very significant difference compared to the blank control group, P < 0.001;
# shows a significant difference compared to preservation solution C, P < 0.05;
# indicates a very significant difference compared to the blank control group, P < 0.001.
The above P values are statistically obtained according to the significance test method.
Therefore, the human menstrual blood preservative fluid can preserve isolated menstrual blood for 48 hours, the survival rate reaches 79.86 +/-5.88%, the technical problem of isolated menstrual blood preservation is solved, and meanwhile, the separated cells are not differentiated, and are stable in quality and high in purity.
Claims (9)
1. A preserving fluid for human menstrual blood is characterized in that the ingredients of the preserving fluid comprise normal saline, hirudin, glucose, hydroxyethyl starch, levofloxacin, vitamin complex, glutathione and necrotizing apoptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor.
2. The preservation solution according to claim 1, wherein the concentration of hirudin is (1 ~ 5). times.hirudin.
3. The preservation solution according to claim 1, wherein the concentration of hydroxyethyl starch is 1 ~ 5%.
4. The preservation solution according to claim 1, wherein the concentration of levofloxacin is (1 ~ 2) x levofloxacin.
5. The preservation solution according to claim 1, wherein the necroptosis inhibitor Necrostatin-1/RIP1 kinase is inhibited at a concentration of 5 ~ 10 ng/mL.
6. The preservation solution according to claim 1, wherein the concentration of the multivitamin is 1 ~ 5 mg/mL.
7. The preservation solution according to claim 1, wherein the concentration of glutathione is 1 ~ 5 mg/mL.
8. A preparation method of human menstrual blood preservative fluid is characterized by comprising the following steps:
1) preparing a solution A: adding glucose, hirudin and levofloxacin into physiological saline to obtain solution A;
2) preparing a solution B: adding hydroxyethyl starch, vitamin complex and glutathione into normal saline to obtain solution B;
3) preparing a solution C: mixing solution A and solution B, adding necroptosis inhibitor Necrostatin-1/RIP1 kinase inhibitor to obtain solution C, filtering with filter membrane, and storing at 4 deg.C.
9. The method for producing a preservative solution according to claim 8, wherein the volume ratio of the solution A to the solution B in the step 3) is 1: 1.
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| CN201910918390.3A CN110622954A (en) | 2019-09-26 | 2019-09-26 | Preservation solution for human menstrual blood and preparation method thereof |
| PCT/CN2019/127785 WO2021056885A1 (en) | 2019-09-26 | 2019-12-24 | Preservation fluid used for human menstrual blood and preparation method therefor |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110278940A (en) * | 2019-07-11 | 2019-09-27 | 河南省农业科学院畜牧兽医研究所 | A kind of Boar spermatozoa preservative agent and its preparation method and application |
| WO2021056885A1 (en) * | 2019-09-26 | 2021-04-01 | 广东华夏健康生命科学有限公司 | Preservation fluid used for human menstrual blood and preparation method therefor |
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- 2019-12-24 WO PCT/CN2019/127785 patent/WO2021056885A1/en active Application Filing
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Application publication date: 20191231 |