WO2021056885A1 - Fluide de conservation utilisé pour le sang menstruel humain et son procédé de préparation - Google Patents

Fluide de conservation utilisé pour le sang menstruel humain et son procédé de préparation Download PDF

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Publication number
WO2021056885A1
WO2021056885A1 PCT/CN2019/127785 CN2019127785W WO2021056885A1 WO 2021056885 A1 WO2021056885 A1 WO 2021056885A1 CN 2019127785 W CN2019127785 W CN 2019127785W WO 2021056885 A1 WO2021056885 A1 WO 2021056885A1
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Prior art keywords
preservation solution
liquid
preservation
preparation
menstrual blood
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PCT/CN2019/127785
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English (en)
Chinese (zh)
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李文东
宋云庆
黎波
卢瑞珊
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广东华夏健康生命科学有限公司
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Publication of WO2021056885A1 publication Critical patent/WO2021056885A1/fr

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    • 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

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  • the invention belongs to the biological technology field of stem cell preservation, and specifically relates to a human menstrual blood preservation liquid and a preparation method thereof.
  • Menstruation is regulated by the interaction between the reproductive hormones of the hypothalamus, pituitary gland and ovary. During the menstrual and proliferative phases of the menstrual cycle, the level of estradiol and progesterone in the blood is very low. The negative feedback effect of the thalamus is weakened or eliminated, leading to an increase in the secretion of hormones that promote gonadal hormone release by the hypothalamus, which in turn leads to an increase in follicle stimulating hormone and luteinizing hormone secreted by the adenohypophysis, thereby causing follicles to develop and estrogen secretion gradually increased. At this time, estrogen stimulates the endometrium to enter the proliferation phase.
  • Luteinizing hormone increases the secretion of progesterone, leading to ovulation.
  • the levels of estrogen and progesterone increased. This has the effect of strengthening the negative feedback inhibition on the hypothalamus and the pituitary gland, thus reducing the levels of ovulation stimulating hormone and luteinizing hormone, leading to the degeneration of the corpus luteum, and then reducing the levels of estrogen and progesterone.
  • the endometrium loses the support of these two hormones, peels off and bleeds, and menstruation occurs. At this time, the reduction of estrogen and progesterone starts the next menstrual cycle.
  • menstruation The main components of menstruation are blood (3/4 arterial blood, 1/4 venous blood), endometrial tissue fragments and various active enzymes and biological factors. Among them, fibrinolytic enzyme makes menstrual blood liquid and does not cause coagulation. Prostaglandins Play the role of contraction of the uterus. Menstrual blood coagulates rapidly in a short period of time in vitro, and its vitality decreases, which directly affects the cell quality of endometrial stem cells in menstrual blood. The viability of endometrial stem cells is directly related to the preservation of menstrual blood. The effect of menstrual blood clotting and hormones in menstrual blood on cell quality after in vitro; how to avoid the above has become the primary problem to be solved.
  • the menstrual preservation solution in the prior art generally adds penicillin, amino acids, human albumin and other substances to preserve isolated tissues, but due to its single nutrient composition, high cost, high concentration of antibiotics, etc., although antibiotics can reduce contamination
  • antibiotics can reduce contamination
  • there is a certain degree of toxicity to placental stem cells resulting in low viability and even death of seed cells during long-term storage or transportation.
  • several amino acids and antibiotics were added to the buffer solution composed of inorganic ions to maintain the viability of the cells in vitro and reduce the infection rate.
  • the purpose of the present invention is to provide a preservation solution for human menstrual blood and a preparation method thereof, and to provide a method that can maintain the viability of menstrual-derived endometrial stem cells without causing cell functional variation.
  • the preservation solution In the prior art, albumin is often used as a source of nutrition for maintaining the vitality of placental mesenchymal stem cells.
  • the present invention further improves the ion buffer system and nutritional system of the preservation solution, while optimizing the composition of the ingredients in the preparation and improving the transportation. Conditions, as much as possible to maintain the survival rate of cells and maintain their biological characteristics.
  • the present invention provides a preservation solution for human menstrual blood.
  • the components of the preservation solution include physiological saline, hirudin, glucose, hydroxyethyl starch, levofloxacin, multivitamins, glutathione, and necrostatin, an inhibitor of necroptosis -1/RIP1 kinase inhibitor.
  • the concentration of the hirudin is (1-5) ⁇ hirudin.
  • Hirudin is the most active and the most studied ingredient among the various active ingredients extracted from leech and its salivary glands. It is a small molecule protein (polypeptide) composed of 65-66 amino acids. Hirudin has a strong inhibitory effect on thrombin and is the strongest natural and purposeful thrombin inhibitor found so far.
  • the physiological saline-based buffer solution purchased from a reagent company, can ensure the stability of the preservation solution buffer system and avoid quality differences caused by artificial configuration.
  • the glucose can be directly absorbed and utilized by the cells, which improves the resistance ability of the cells and maintains the activity of the cells.
  • the concentration of the hydroxyethyl starch is 1 to 5%, which can maintain the osmotic pressure of the preservation solution, so that the endometrial stem cells can be stored in an isotonic and isostatic environment for the activity of placental mesenchymal stem cells.
  • the concentration of the levofloxacin is (1-2) ⁇ levofloxacin.
  • Levofloxacin is one of the quinolone drugs. It has a broad-spectrum antibacterial effect and strong antibacterial effect. It can effectively inhibit various types of female vaginal flora.
  • Enterobacteriaceae bacteria such as Escherichia coli, Klebsiella, Proteus, Salmonella, Shigella and Haemophilus influenzae, Legionella pneumophila, Neisseria gonorrhoeae and other gram-negative bacteria have strong antibacterial activity; against Staphylococcus aureus, Streptococcus pneumoniae, and purulent Gram-positive bacteria such as Streptococcus vulgaris and Mycoplasma pneumoniae and Chlamydia pneumoniae also have antibacterial effects.
  • the concentration of the necrostatin-1/RIP1 kinase inhibitor is 5-10 ng/mL, and the necrostatin-1/RIP1 kinase inhibitor can directly keep cell apoptosis and preserve the biological Features, can improve the survival rate of cells in an all-round way, and maintain cell survival.
  • the concentration of the multivitamin is 1-5 mg/mL
  • the concentration of the glutathione is 1 to 5 mg/mL.
  • Glutathione is a powerful antioxidant. It exists in every cell of the body and can remove excess free radicals to achieve antioxidant effects.
  • the present invention also provides a method for preparing human menstrual blood preservation solution, which includes the following steps:
  • liquid A 1) Preparation of liquid A: adding glucose, hirudin and levofloxacin to physiological saline to obtain liquid A;
  • liquid B Preparation of liquid B: add hydroxyethyl starch, multivitamins and glutathione to physiological saline to obtain liquid B.
  • the combined use of multivitamins and glutathione can improve the antioxidant function of cells in an all-round way;
  • Step 3 The volume ratio of the mixing of liquid A and liquid B is 1:1, and the pore size of the filter membrane is 0.22 ⁇ m.
  • the preservation solution for human menstrual blood of the present invention uses glucose as the basic nutrient component, physiological saline as a buffer solution, and also contains different anti-apoptotic agents, which can enhance the resistance of cells, and a suitable preservation environment and nutrient supply can be very effective. It maintains the vitality of menstrual-derived endometrial stem cells, ensures the long-term survival rate of the isolated placenta in the preservation solution, does not cause cell functional variation, and has anticoagulant and antibacterial effects on isolated menstruation.
  • the preservation solution of the present invention can preserve the menstrual blood in vitro for 48 hours, can maintain the original biological characteristics of endometrial stem cells within 48 hours, maintain the quality of the cells, and solve the technical problem of in vitro preservation of menstrual blood. The separated cells have not differentiated, the cell quality is stable, the purity is high, and it can be produced on a large scale.
  • the preparation steps of the preservation solution of the present invention are simple, can be configured in a large amount in a short time, have low cost, stable components, safety, and no toxic and side effects.
  • Figure 1 is a flow cytometric identification diagram of a control group in which the preservation solution of the present invention is used to extract endometrial stem cells after menstrual blood;
  • Fig. 2 is a flow cytometric identification diagram of a sample group in which the preservation solution of the present invention is used to extract endometrial stem cells after menstrual blood;
  • Fig. 3 is a cell morphology detection diagram of the preservation solution of the present invention for extracting endometrial stem cells after menstrual blood.
  • all the raw materials and reagents of the present invention are the raw materials and reagents in the conventional market.
  • a method for preparing a preservation solution for human menstrual blood :
  • liquid A Preparation of liquid A: add glucose, hirudin and levofloxacin to 0.9% saline to obtain liquid A, and obtain 4% glucose, (2 ⁇ 10) x hirudin, (2 ⁇ 4) x levofloxacin;
  • liquid B Preparation of liquid B: add hydroxyethyl starch, multivitamins and glutathione to 0.9% normal saline to obtain liquid B, obtain 2-10% hydroxyethyl starch, 2-10mg/mL multivitamin, 2 ⁇ 10mg/mL glutathione;
  • the menstrual blood of women aged 18-36 with regular menstrual cycles and menstrual periods of 5-7 is used as the experimental object. Menstruation starts with redness as the starting point.
  • the menstrual blood is collected within 12-36 hours, and the vulva is wiped with sterile gauze before collection. , Put the menstrual cup into the vagina, take it out for 15-30 minutes, suck out the menstrual blood with a sterile straw, mix it gently and thoroughly with the preservation solution prepared in the example at a volume ratio of 1:10, and store it at 4°C;
  • the collected menstrual blood enters the cell separation culture within 0h, 24h, and 48h, and the preservation process is kept at 4°C;
  • menstrual blood volume ratio 1:1; use a disposable pipette to add lymphocyte separation fluid to each tube, and use a disposable pipette or Pasteur pipette to slowly transfer the diluted blood to the lymphocyte separation fluid The surface, so that a clear interface between the two. Transfer the centrifuge tube to a centrifuge and centrifuge at 700g for 15 minutes. After centrifugation, the albuginea layer was sucked out, transferred to another 50mL centrifuge tube, RPMI 1640 medium was added to 40mL with a disposable pipette, the PBMC was resuspended, and the PBMC was centrifuged at 500g for 5min.
  • the supernatant was discarded, and the cell pellet was resuspended with high-sugar DMEM+10% FBS, inoculated into a 10 cm dish, and transferred to a 37°C, 5.0% CO 2 , humidity 95.0% CO 2 incubator for culture.
  • Morphological examination Take the endometrial stem cells in the logarithmic growth phase and observe the morphology of the cells under an inverted microscope.
  • Proliferation rate 48 hours after cell inoculation, change the medium; take a dish of cells every 48 hours for enzymatic hydrolysis until the confluence of the batch of cells reaches 100%, count the number of cells, and get the cell proliferation rate.
  • Flow cytometry results detection Take endometrial stem cells in logarithmic growth phase, adjust the cell density to 1 ⁇ 10 6 cells, and take anti-human CD105, CD90, CD73, CD45, CD34, HLA-DR, respectively Add 2.5 ⁇ L of each of the monoclonal antibodies to the cell suspension, and incubate at room temperature for 20 minutes in the dark, as a sample group, and set up an isotype control as a control group. Centrifuge at 1500r/min for 5 minutes, discard the supernatant, and use PBS containing 10% FBS. Wash twice, resuspend with 500 ⁇ L of 1640, and test on the machine (the sample group contains fluorescent antibodies, and the gate set by the isotype control is used as the limit to determine the specific purity of the cells);
  • Figure 1 is the flow cytometric identification diagram of the control group
  • Figure 2 is the flow cytometry identification diagram of the sample group. From the flow cytometry results, the endometrium stem cells highly express CD73, CD90, and CD105, and the expression rates are 99.9% and 99.9%, respectively. , 97.2%; low expression of HLA-DR, CD45, CD19, CD11b, CD34, the expression rates were 0.1%, 0.0%, 0.1%, 0.4%, 0.2%.
  • mesenchymal stem cells meet the surface antigen characteristics of mesenchymal stem cells (identification criteria for mesenchymal stem cells promulgated by the International Stem Cell Association ISCT: positive expression: CD73, CD90, CD105>95.0%; negative expression of HLA-DR, CD45, CD11b, CD34, CD19 ⁇ 2.0%), indicating that the endometrial stem cells have not differentiated and still maintain the characteristics of stem cells.
  • Example 1 Take the preservation solution prepared in Example 1 and store the menstrual blood according to Test Example 2. After the extracted endometrial stem cells are expanded and cultured, 3 samples (sample 1, sample 2, sample 3) are taken for cell viability In addition to morphological detection, the cell viability detection is shown in the following table (Table 1). It can be seen that the preservation solution of this example has a significant preservation effect on cells.
  • Figure 3 shows the morphological detection of the cells.
  • the endometrial stem cells were tested by trypan blue. The cells were all rejected, the dead cells were stained blue, and the living cells were rejected, indicating the preservation of the endometrial cells in this example. It still maintains vitality under the action of liquid.
  • Blank control group 0.9% normal saline, 2 ⁇ levofloxacin;
  • Preservation solution A 0.9% saline containing 1 ⁇ hirudin, 2% glucose, 2% hydroxyethyl starch, 2 ⁇ levofloxacin;
  • Preservation solution B 0.9% saline containing 1 ⁇ Hirudin, 2% glucose, 2% hydroxyethyl starch, 2 ⁇ levofloxacin, 5mg/mL multivitamin, 5mg/mL glutathione;
  • Preservation solution C 0.9% saline containing 1 ⁇ hirudin, 2% glucose, 2% hydroxyethyl starch, 2 ⁇ levofloxacin, 5mg/mL multivitamin, 5mg/mL glutathione, 10ng/mL necrotic apoptosis Death inhibitor Necrostatin-1/RIP1 kinase inhibitor.
  • the above P value is statistically based on the significance test method.
  • the human menstrual blood preservation solution of the present invention can preserve the menstrual blood in vitro for as long as 48 hours, which solves the technical problem of in vitro preservation of menstrual blood.
  • the separated cells have not differentiated, and the cell quality is stable and the purity is stable. high.

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Abstract

La présente invention appartient au domaine biotechnologique de la conservation de cellules souches. L'invention concerne un fluide de conservation utilisé pour le sang menstruel humain et son procédé de préparation. Les ingrédients du fluide de conservation comprennent une solution saline normale, de l'hirudine, du glucose, de l'amidon d'hydroxyéthyle, de la lévofloxacine, des multivitamines, du glutathion et un inhibiteur de nécroptose, la Nécrostatine 1/inhibiteur de la kinase RIP1. Le procédé de préparation du fluide de conservation comprend les étapes consistant à : 1) préparer une solution A : ajouter respectivement du glucose, de l'hirudine et de la lévofloxacine dans une solution saline normale ; 2) préparer une solution B : ajouter respectivement de l'amidon d'hydroxyéthyle, des multivitamines et du glutathion dans la solution saline normale ; et 3) préparer une solution C : mélanger la solution A et la solution B, puis ajouter l'inhibiteur de nécroptose, la Nécrostatine 1/inhibiteur de la kinase RIP1, et après filtration à l'aide d'une membrane filtrante, stocker à 4 °C pour une utilisation ultérieure. Le liquide de conservation de la présente invention peut maintenir les caractéristiques biologiques d'origine des cellules souches endométriales pendant 48 heures, la qualité des cellules est stable, et la pureté est élevée.
PCT/CN2019/127785 2019-09-26 2019-12-24 Fluide de conservation utilisé pour le sang menstruel humain et son procédé de préparation WO2021056885A1 (fr)

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CN110278940A (zh) * 2019-07-11 2019-09-27 河南省农业科学院畜牧兽医研究所 一种猪精子保存剂及其制备方法和应用
CN110622954A (zh) * 2019-09-26 2019-12-31 广东华夏健康生命科学有限公司 一种用于人月经血的保存液及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105532647A (zh) * 2016-02-03 2016-05-04 广州赛莱拉干细胞科技股份有限公司 经血保存液及其应用、经血源宫内膜干细胞的分离培养方法
CN106011052A (zh) * 2016-06-15 2016-10-12 浙江奥比特生物科技有限公司 一种高纯度经血来源干细胞的制备方法
KR20180088068A (ko) * 2017-01-26 2018-08-03 중앙대학교 산학협력단 네크로스타틴-1을 포함하는 정원줄기세포의 동결보존용 조성물 및 이를 이용한 정원줄기세포의 동결보존 방법
CN109845727A (zh) * 2019-03-25 2019-06-07 西安医斯美生物科技有限公司 经血保存液及经血来源间充质干细胞冻存方法
CN110622954A (zh) * 2019-09-26 2019-12-31 广东华夏健康生命科学有限公司 一种用于人月经血的保存液及其制备方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103966162B (zh) * 2014-05-29 2016-07-06 成都清科生物科技有限公司 一种经血来源间充质干细胞分离方法
WO2016045022A1 (fr) * 2014-09-25 2016-03-31 深圳华大基因科技有限公司 Solution de conservation de prélèvement de sang périphérique maternel et procédé de conservation d'un prélèvement de sang périphérique maternel
CN105687240B (zh) * 2016-03-14 2019-05-07 广州赛莱拉干细胞科技股份有限公司 一种经血源干细胞制剂及其制备方法与应用
CN107251894A (zh) * 2017-06-30 2017-10-17 太仓东浔生物科技有限公司 一种高存活率的细胞冻存方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105532647A (zh) * 2016-02-03 2016-05-04 广州赛莱拉干细胞科技股份有限公司 经血保存液及其应用、经血源宫内膜干细胞的分离培养方法
CN106011052A (zh) * 2016-06-15 2016-10-12 浙江奥比特生物科技有限公司 一种高纯度经血来源干细胞的制备方法
KR20180088068A (ko) * 2017-01-26 2018-08-03 중앙대학교 산학협력단 네크로스타틴-1을 포함하는 정원줄기세포의 동결보존용 조성물 및 이를 이용한 정원줄기세포의 동결보존 방법
CN109845727A (zh) * 2019-03-25 2019-06-07 西安医斯美生物科技有限公司 经血保存液及经血来源间充质干细胞冻存方法
CN110622954A (zh) * 2019-09-26 2019-12-31 广东华夏健康生命科学有限公司 一种用于人月经血的保存液及其制备方法

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