CN109481466B

CN109481466B - Methods and cell preparations for treating premature ovarian failure using placental mesenchymal stem cells

Info

Publication number: CN109481466B
Application number: CN201811568010.XA
Authority: CN
Inventors: 许晓椿; 李容; 肖海蓉; 刘冰
Original assignee: BOYALIFE Inc
Current assignee: BOYALIFE Inc
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2021-02-05
Anticipated expiration: 2038-12-21
Also published as: CN109481466A

Abstract

The invention relates to a method and a cell preparation for treating premature ovarian failure by using placenta mesenchymal stem cells. Specifically, one aspect of the present invention relates to a cell preparation which is a cell suspension prepared by suspending mesenchymal stem cells such as placental mesenchymal stem cells in a 0.9% sodium chloride solution, the cell preparation being prepared by a method comprising: and transferring the mesenchymal stem cells obtained by cell passage to a centrifuge tube, centrifuging, discarding supernatant, adding 0.9% sodium chloride solution for resuspension, and thus obtaining the cell preparation. The mesenchymal stem cell is prepared by a method comprising the following steps: processing the placenta lobules, digesting and terminating the mixed enzyme, collecting primary cells, freezing and storing the primary cells, recovering the cells, carrying out cell passage, detecting the cells, freezing and storing the cells, and correlating the cells with a database. The cell preparation prepared by the invention has excellent biological effect on treating premature ovarian failure.

Description

Methods and cell preparations for treating premature ovarian failure using placental mesenchymal stem cells

Technical Field

The invention belongs to the field of biotechnology and biomedicine, and relates to a method for treating Premature Ovarian Failure (POF) by using stem cells and a cell preparation used in the method. In particular, the present invention relates to the isolation of stem cells from placenta, and more particularly, to the isolation of mesenchymal stem cells from placenta and the use of such placental mesenchymal stem cells for the treatment of premature ovarian failure, and even more particularly, to the isolation and culture of mesenchymal stem cells from placenta tissue into mesenchymal stem cells using a digestive enzyme composition having a unique formulation according to the present invention, and the use of such placental mesenchymal stem cells for the treatment of premature ovarian failure. The method can effectively improve the efficiency of separating the mesenchymal stem cells from the placenta, and further can effectively improve the effect of treating premature ovarian failure by using the mesenchymal stem cells of the placenta.

Background

Premature Ovarian Failure (POF) refers to the phenomenon of amenorrhea before the age of 40 due to ovarian failure. Is a disease characterized by amenorrhea, infertility, estrogen deficiency, follicular decrease and gonadotropin elevation, accompanied by a range of low estrogen symptoms such as: hot flashes, profuse sweating, flushing of the face, low libido and the like seriously affect the physical and mental health of women. In addition, women with POF have an increased risk of osteoporosis, cardiovascular disease and dementia. POF accounts for 1 to 3% of women, and the incidence of POF has been on the rise in recent years.

POF has a complex etiology, is not completely elucidated, but is associated with autoimmune response, infection, genetic factors, chemotherapy, radiotherapy, surgery, and other therapeutic effects, and endocrine dysfunction. Currently, the most common therapeutic method for POF is Hormone Replacement Therapy (HRT). Although the treatment has a certain relieving effect on the clinical symptoms of POF, HRT cannot fundamentally repair damaged ovaries and recover the ovarian function. In addition, studies have shown that long-term HRT treatment increases the risk of heart disease and stroke, and may increase the risk of breast cancer and autoimmune disease. Therefore, new therapeutic strategies are needed to restore ovarian function in POF patients.

Treatments based on stem cells, particularly mesenchymal stem cells, have created new hopes for POF patients to improve their ovarian function and restore fertility.

Mesenchymal Stem Cells (MSCs), such as human mesenchymal stem cells, were first isolated from bone marrow and a class of tissue stem cells derived from the mesoderm, which have the potential for multipotent differentiation and the ability to self-renew, have the ability to differentiate into various adult cells, such as osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, hepatocytes, etc., under specific conditions in vivo and in vitro (Cap AI. mesenchymal stem cells. J Orthop Res.1991,9:641-650.Pittenger MF, Mackay AM, Beck, et al. multilineage patent of epithelial man stem cells. science.1999; 284:143 Across 147). Recent research shows that the mesenchymal stem cells have the functions of immunoregulation and hematopoietic support, and are easy to introduce and express exogenous genes. Therefore, the mesenchymal stem cells are not only seed cells in the construction of tissue engineering bone, cartilage and cardiac muscle and important carrier cells in gene therapy, but also have wide application prospect in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft-versus-host reaction. Mesenchymal stem cells have the characteristic of adherent growth in vitro, and by utilizing the characteristic, the mesenchymal stem cells are successfully separated and cultured from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like.

At present, the reported mesenchymal stem cells are mainly derived from bone marrow and are obtained by adopting a density gradient centrifugation method. Although the separation method is simple, the donor needs to undergo a painful operation for taking marrow, and has a high infection chance in and after the material is taken; because the content of MSC in human bone marrow is very rare, every 10⁵～10⁶Only about 1 of the mononuclear cells are present, and the number, proliferation and differentiation capacity of mesenchymal stem cells in the bone marrow are remarkably reduced with the increase of the age, so that the research and application, particularly the clinical application of the mesenchymal stem cells are limited. The placenta, which originates from the extraembryonic mesoderm during the embryonic development, is composed of mesenchyme, blood vessels and trophoblasts, and contains a large amount of mesenchyme components. Recent research shows that the placenta contains abundant stem cells, and the separation and culture of the pluripotent stem cells from the placenta opens up a brand-new and abundant source for experimental research and clinical application.

The existing methods for establishing a placental stem cell bank by isolating stem cells from placenta have many disadvantages, such as insufficient purity and/or low quantity, and thus show that these methods are not satisfactory. For example, CN101270349A (chinese patent application No. 200810061267.6, published 2008/9/24) discloses an invention entitled "placental mesenchymal stem cell isolation and in vitro expansion culture method"; CN101693884A (chinese patent application No. 200910117522.9, published 2010, 4 months and 14 days) entitled "a method for separating and extracting stem cells from placenta, umbilical cord or adipose tissue"; CN102146359A (chinese patent application No. 201110005964.1, published 2011/8/10) discloses an invention entitled "method for extracting original mesenchymal stem cells from placenta and serum-free expansion". In addition, chinese patent application No. 201210044648X discloses a method of isolating mesenchymal stem cells from placenta. These processes are to be further improved in terms of purity and/or recovery of the extract. In addition, an invention publication CN107299082A (chinese patent application No. 201710653583.1, published 2018, 10 months and 27 days) by the present inventor team describes a method of obtaining placental mesenchymal stem cells that has been shown to exhibit some excellent properties.

There remains a need in the art for new methods for isolating stem cells from placenta, and in particular for methods that efficiently isolate mesenchymal stem cells from placenta. In addition, there remains a need in the art for new digestive enzyme compositions for use in methods of isolating mesenchymal stem cells from placenta in an effort to improve the efficiency of the methods of isolating mesenchymal stem cells from placenta. In addition, there remains a need in the art for new methods for treating premature ovarian failure, and in particular, new more effective methods for treating premature ovarian failure using mesenchymal stem cells are expected.

Disclosure of Invention

Objects of the invention include one or more of the following: on one hand, the method solves the defects of the existing method for obtaining the placenta mesenchymal stem cells, provides a practical, simple and efficient method for separating the mesenchymal stem cells from the placenta tissues and culturing the mesenchymal stem cells into the mesenchymal stem cells and optionally establishing a placenta stem cell bank; in another aspect, a digestive enzyme composition is provided for the above method of isolating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells; in yet another aspect, a cell preparation is provided for treating premature ovarian failure using mesenchymal stem cells. The inventor finds that by adopting a special operation method and a special formula of the digestive enzyme composition, the obtained cells have high purity and/or high cell recovery rate, and the specific cell preparation can be more effectively used for treating premature ovarian failure. The present invention has been completed based on such findings.

Accordingly, in a first aspect the invention provides a cell preparation, e.g. useful for the treatment of premature ovarian failure, which is a cell suspension formulated by suspending mesenchymal stem cells (e.g. placental mesenchymal stem cells) in a 0.9% sodium chloride solution.

The cell preparation according to the first aspect of the present invention, wherein the cell concentration is 1 to 10X 10⁶One cell/ml, e.g., 1-5X 10⁶Individual cells/ml, e.g. 1-3X 10⁶Individual cells/ml.

According to the cell preparation of the first aspect of the present invention, magnesium gluconate and phospholipids are further added to the 0.9% sodium chloride solution. In one embodiment, the magnesium gluconate is added in an amount to provide a magnesium ion concentration of 5 mmol/L. In one embodiment, the phospholipid is added at a concentration of 0.2 mg/ml. In one embodiment, the phospholipid is of injection grade soy origin. It has been surprisingly found that the biological effect of the cell preparation of the present invention in treating premature ovarian failure can be significantly improved by simultaneously adding a small amount of magnesium salt and phospholipid. The present inventors have also found that when only a magnesium salt, or only a phospholipid, or the above-mentioned magnesium salt is replaced with another salt such as a calcium salt, a zinc salt, a copper salt, or the like, is added to the cell preparation of example 4 of the present invention, the results are far inferior to those of the PD-MSC group a in which a magnesium salt and a phospholipid are added simultaneously, and the results in these cases are similar to those of the PD-MSC group b of example 4 of the present invention.

A cell preparation according to the first aspect of the invention, which is prepared by a method comprising: and transferring the mesenchymal stem cells obtained by cell passage to a centrifuge tube, centrifuging, discarding supernatant, adding 0.9% sodium chloride solution for resuspension, and thus obtaining the cell preparation.

The cell preparation according to the first aspect of the present invention, wherein the mesenchymal stem cells are prepared by a method comprising the steps of:

(1) treatment of placental leaflets: placing placenta in a white porcelain dish, washing with tissue cleaning solution to remove blood stasis of placenta, shearing 20g of placenta lobule tissue in a steel cup, cleaning twice with the tissue cleaning solution, soaking for 5min, and weighing 15g of better tissue in a 100mm glass dish; adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm³About the size, 100ml of tissue cleaning fluid is added, the mixture is stirred evenly and filtered by a 300-mesh filter screen, and the operation is repeated to clean the tissue cleaning fluid twice so as to remove blood cells;

[ wherein the tissue cleansing solution is 0.9% physiological saline containing 1% double antibody ]

(2) Mixed enzyme digestion and termination: adding the cleaned leaflet tissue into 15-30 ml (such as 20-25 ml, such as 23ml) of mixed enzyme digestive juice preheated at 37 ℃, fully mixing uniformly, then digesting for 30min by shaking at the temperature of 37 ℃ and 100rpm by using a shaking table, and after digestion is finished, adding 2ml of FBS into the tissue juice to stop digestion;

[ wherein the mixed enzyme digestive juice contains: 15-30 volumes of Hank ' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme, 0.2-2 volumes of DNA type I enzyme (e.g., 20-25 volumes of Hank ' S balanced salt solution, 0.3-0.5 volumes of Liberase MNP-S enzyme, 0.5-1 volumes of DNA type I enzyme, e.g., 22 volumes of Hank ' S balanced salt solution, 0.4 volumes of Liberase MNP-S enzyme, 0.7 volumes of DNA type I enzyme); the Liberase MNP-S enzyme is for example the Liberase MNP-S enzyme from Roche, for example from Siberian organisms, the cat # of which: 5578582001]

(3) Collecting primary cells: adding 50ml of tissue cleaning fluid into the tissue fluid obtained in the last step, uniformly mixing, filtering by a 300-mesh sieve, and collecting cell fluid; washing the digested tissue twice repeatedly, combining the filtrates of the two times into a centrifuge tube, and centrifuging at 1500rpm for 8min (acceleration 9 and deceleration 7); removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, centrifuging at 1500rpm for 8min (acceleration 9, deceleration 7); removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells;

[ cell suspension of this primary cell can be subjected to cell counting using a sysmex hematology analyzer ]

(4) Freezing and storing primary cells: centrifuging the cell suspension at 1800rpm for 10min (acceleration 9 and deceleration 7), collecting cell precipitate and supernatant 5ml, re-suspending, slowly adding frozen stock solution 10ml, and shaking; subpackaging the obtained cell suspension into 9 freezing tubes of 2ml, each tube of 1.5ml, placing in a precooled program cooling box, performing program cooling by using a program cooling instrument, and transferring the cells into a liquid nitrogen storage tank for freezing;

[ wherein, the formula of the freezing solution is as follows: 65% DMEM-F12, 15% Human Serum Albumin (HSA), 20% DMSO, e.g. WAK brand DMSO ]

(5) Cell recovery: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; centrifuging at 1200rpm for 5min (acceleration 9, deceleration 7), removing supernatant, and adding 5ml complete culture medium for resuspension; inoculating each tube of cells into 1T 75 culture bottle, supplementing complete culture medium to 30ml, and culturing in CO2 incubator (37 deg.C, 5% CO2, saturated humidity); every 3-4 days, using complete culture mediumThe liquid is changed again, and counted according to the clone formation after recovery for 12 days until the cell density is not less than 3000 cells/cm²The following passages can be performed;

[ wherein, the complete medium is DMEM-F12 medium containing 10% FBS ]

(6) Cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of the cells fall off, adding 5ml complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9, deceleration 7), discarding supernatant, adding 5ml complete culture medium for resuspension, counting, and inoculating to a culture bottle, wherein the cell density is 8000-12000 cells/cm²Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation; repeating the operations in sequence to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain the mesenchymal stem cells of each generation.

The cell preparation according to the first aspect of the present invention, in the preparation of mesenchymal stem cells, further comprises:

(7) aiming at the placenta mesenchymal stem cells obtained in the step (6), detecting at least one item of the following items: cell viability, cell contamination, genetic disease, HLA-ABC/DR match.

(8) and (4) freezing and storing the passage-generated placenta mesenchymal stem cells obtained in the step (6) in liquid nitrogen.

(9) establishing a database of placental stem cells comprising the above information, and correlating the database with the cryopreserved cells of step (8).

The cell preparation according to the first aspect of the present invention, wherein the cell purity of each generation of the placental mesenchymal stem cells obtained is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

The cell preparation according to the first aspect of the invention wherein said Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. The cell preparation according to the first aspect of the invention, wherein the mixed enzyme digestive juice contains Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme, and 0.2-0.3 g/L zinc chloride. It has been surprisingly found that with the use of mixed enzyme digests with added zinc chloride in this concentration range, the resulting primary cells have greater than 60% CD73 expression, no CD45 expression, and mesenchymal stem cell content in the resulting primary cells of 60% -70%, showing extremely high stem cell concentrations; when the zinc chloride is not added into the mixed enzyme digestive juice, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually within the range of 31-38 percent.

The cell preparation according to the first aspect of the present invention, wherein said cell activity measurement is counting the number of viable cells before and after cryopreservation using trypan blue staining method.

The cell preparation according to the first aspect of the present invention, wherein said cell contamination detection detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B, hepatitis C, HIV, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.

The cell preparation according to the first aspect of the present invention, wherein said detection of the genetic disease is a method of detecting the presence or absence of the genetic disease in the cryopreserved cells by using molecular genetics.

A cell preparation according to the first aspect of the invention, wherein said HLA-ABC/DR match is a test cell for HLA-ABC/DR phenotype.

The cell preparation according to the first aspect of the present invention, wherein said placental mesenchymal stem cells are frozen in liquid nitrogen by a temperature-programmed process.

A cell preparation according to the first aspect of the invention, wherein the database includes data relating to all of the cells stored, including but not limited to: the biological characteristic test result of the cell, the identification result of the multi-directional differentiation potential, the molecular genetic diagnosis result of the cell, and the detailed information of the fetus and the parents thereof.

Further, the present invention provides in a second aspect a method for isolating mesenchymal stem cells from placental tissue and culturing the mesenchymal stem cells into mesenchymal stem cells, the method comprising the steps of:

(5) Cell recovery: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; centrifuging at 1200rpm for 5min (acceleration 9, deceleration 7), removing supernatant, and adding 5ml complete culture medium for resuspension; inoculating each tube of cells into 1T 75 culture bottle, supplementing complete culture medium to 30ml, and culturing in CO2 incubator (37 deg.C, 5% CO2, saturated humidity); performing total liquid change every 3-4 days with complete culture medium, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm²The following passages can be performed;

[ wherein, the complete medium is DMEM-F12 medium containing 10% FBS ]

(6) Cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of cells are shed, addingTerminating digestion with 5ml of complete medium, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9, deceleration 7), discarding the supernatant, adding 5ml of complete medium for resuspension, counting, and inoculating to a culture flask with cell density of 8000-12000 cells/cm²Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation; repeating the operations in sequence to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain the mesenchymal stem cells of each generation.

The method according to any embodiment of the second aspect of the present invention, further comprising:

The method according to any embodiment of the second aspect of the present invention, wherein the cell purity of the obtained generations of placental mesenchymal stem cells is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

The method according to any embodiment of the second aspect of the present invention wherein said Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. The method according to any embodiment of the second aspect of the present invention, wherein the mixed enzyme digestive juice contains Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA I type enzyme, and 0.2-0.3 g/L zinc chloride. It has been surprisingly found that with the use of mixed enzyme digests with added zinc chloride in this concentration range, the resulting primary cells have greater than 60% CD73 expression, no CD45 expression, and mesenchymal stem cell content in the resulting primary cells of 60% -70%, showing extremely high stem cell concentrations; when the zinc chloride is not added into the mixed enzyme digestive juice, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually within the range of 31-38 percent.

The method according to any embodiment of the second aspect of the present invention, wherein said cell viability assay is counting the number of viable cells before and after cryopreservation using trypan blue staining.

The method according to any embodiment of the second aspect of the present invention, wherein said cell contamination detection detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B, hepatitis C, HIV, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.

The method according to any embodiment of the second aspect of the present invention, wherein said detection of genetic disease is a method of detecting the presence of genetic disease in cryopreserved cells using molecular genetics.

A method according to any one of the embodiments of the second aspect of the invention, wherein said HLA-ABC/DR matching is the detection of the HLA-ABC/DR phenotype of the cell.

The method according to any one of the embodiments of the second aspect of the present invention, wherein said placental mesenchymal stem cells are frozen in liquid nitrogen via a temperature-programmed process.

A method according to any embodiment of the second aspect of the invention, wherein the database includes data relating to all of the cells stored, including but not limited to: the biological characteristic test result of the cell, the identification result of the multi-directional differentiation potential, the molecular genetic diagnosis result of the cell, and the detailed information of the fetus and the parents thereof.

Furthermore, in the method of the second aspect of the present invention, there is provided a placental mesenchymal stem cell. Thus in a third aspect the invention provides a placental mesenchymal stem cell.

The placental mesenchymal stem cells according to the third aspect of the invention, wherein the cell purity of each generation of placental mesenchymal stem cells is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

The placental mesenchymal stem cells according to the third aspect of the present invention are prepared by a method comprising the steps of:

[ wherein, the complete medium is DMEM-F12 medium containing 10% FBS ]

(6) Cell passage: the P0 generation cells were washed with PBS and 2ml of pancreas was addedEnzyme digestion for 2-5min until most of cells fall off, adding 5ml of complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9, deceleration 7), discarding supernatant, adding 5ml of complete culture medium to resuspend, counting, and inoculating to a culture flask, wherein the cell density is 8000-12000 cells/cm²Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation; repeating the operations in sequence to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain the mesenchymal stem cells of each generation.

The placental mesenchymal stem cells according to the third aspect of the present invention, in the preparing step, further comprise:

According to the placental mesenchymal stem cells of the third aspect of the present invention, in the preparing step, the cell purity of each generation of placental mesenchymal stem cells is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

The placental mesenchymal stem cells according to the third aspect of the invention, in the preparing step, the Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. According to the placental mesenchymal stem cell of the third aspect of the present invention, in the preparation step, the mixed enzyme digestion solution further comprises 0.2-0.3 g/L zinc chloride in addition to Hank' S balanced salt solution, Liberase MNP-S enzyme, and DNA type I enzyme. It has been surprisingly found that with the use of mixed enzyme digests with added zinc chloride in this concentration range, the resulting primary cells have greater than 60% CD73 expression, no CD45 expression, and mesenchymal stem cell content in the resulting primary cells of 60% -70%, showing extremely high stem cell concentrations; when the zinc chloride is not added into the mixed enzyme digestive juice, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually within the range of 31-38 percent.

In the placental mesenchymal stem cells according to the third aspect of the present invention, in the preparing step, the cell viability is detected by counting the number of viable cells before and after cryopreservation using trypan blue staining.

The placental mesenchymal stem cells according to the third aspect of the present invention, in the preparing step, the cell contamination detection detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B, hepatitis C, HIV, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.

In the mesenchymal stem cell of placenta according to the third aspect of the present invention, in the preparing step, the genetic disease is detected by detecting whether the cryopreserved cell has a genetic disease or not by using a molecular genetic method.

The mesenchymal stem cell of placenta according to the third aspect of the present invention, wherein in the preparing step, the HLA-ABC/DR matching is to detect HLA-ABC/DR phenotype of the cell.

In the preparation step, the placental mesenchymal stem cells are frozen in liquid nitrogen through a temperature programming process.

In the preparation step of the placental mesenchymal stem cells according to the third aspect of the present invention, the database includes all relevant data of the preserved cells, including but not limited to: the biological characteristic test result of the cell, the identification result of the multi-directional differentiation potential, the molecular genetic diagnosis result of the cell, and the detailed information of the fetus and the parents thereof.

Further, the fourth aspect of the present invention provides a mixed enzyme digestive fluid used in a method for separating mesenchymal stem cells from placenta tissue and culturing the mesenchymal stem cells, the mixed enzyme digestive fluid comprising Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA type I enzyme.

The mixed enzyme digestive juice according to any embodiment of the fourth aspect of the present invention comprises: 15-30 volumes of Hank ' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme, 0.2-2 volumes of DNA type I enzyme (e.g., 20-25 volumes of Hank ' S balanced salt solution, 0.3-0.5 volumes of Liberase MNP-S enzyme, 0.5-1 volumes of DNA type I enzyme, e.g., 22 volumes of Hank ' S balanced salt solution, 0.4 volumes of Liberase MNP-S enzyme, 0.7 volumes of DNA type I enzyme).

The mixed enzyme digest according to any one of the embodiments of the fourth aspect of the present invention, wherein the Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. The mixed enzyme digest according to any one of the fourth aspect of the present invention, wherein zinc chloride is added in a prescribed amount as described herein in addition to the Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA type I enzyme. It has been surprisingly found that superior technical effects as described in the present invention can be exhibited in the case of using a mixed enzyme digest to which zinc chloride is added in this concentration range.

The mixed enzyme digestive fluid according to any one of the embodiments of the fourth aspect of the present invention, wherein the method for separating mesenchymal stem cells from placenta tissue and culturing into mesenchymal stem cells, comprises the steps of:

[ wherein, the complete medium is DMEM-F12 medium containing 10% FBS ]

The mixed enzyme digestive juice according to any embodiment of the fourth aspect of the present invention, wherein the method for separating mesenchymal stem cells from placenta tissue and culturing into mesenchymal stem cells further comprises:

The mixed enzyme digestion solution according to any embodiment of the fourth aspect of the present invention, wherein the cell activity detection in the method of separating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells is counting the number of viable cells before and after cryopreservation using trypan blue staining method.

The mixed enzyme digestive juice according to any embodiment of the fourth aspect of the present invention, wherein the cell contamination detection in the method of separating mesenchymal stem cells from placenta tissue and culturing into mesenchymal stem cells detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B, hepatitis C, HIV, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.

The mixed enzyme digestion solution according to any embodiment of the fourth aspect of the invention, wherein the genetic disease detection in the method for separating mesenchymal stem cells from placenta tissue and culturing into mesenchymal stem cells is to detect whether the cryopreserved cells have genetic diseases or not by using a molecular genetic method.

The mixed enzyme digest of any embodiment of the fourth aspect of the present invention, wherein said HLA-ABC/DR typing in said method of isolating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells is detecting the HLA-ABC/DR phenotype of the cells.

The mixed enzyme digestion solution according to any embodiment of the fourth aspect of the present invention, wherein the method for separating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells comprises freezing the mesenchymal stem cells in liquid nitrogen through a temperature programming process.

The mixed enzyme digestive fluid according to any embodiment of the fourth aspect of the present invention, wherein the method for separating mesenchymal stem cells from placental tissue and culturing into mesenchymal stem cells comprises in the database all relevant data of the preserved cells, including but not limited to: the biological characteristic test result of the cell, the identification result of the multi-directional differentiation potential, the molecular genetic diagnosis result of the cell, and the detailed information of the fetus and the parents thereof.

Further, according to a fifth aspect of the present invention, there is provided a use of a cell preparation (e.g. a cell preparation according to the first aspect of the present invention) in the manufacture of a medicament for the treatment and/or prevention of premature ovarian failure.

Of the various process steps described above, although specific steps are described in some detail or in language specific to the process steps described in the examples of the following detailed description, those skilled in the art will be able to fully appreciate the above-described process steps from the detailed disclosure of the invention as a whole.

Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

In the present invention, the term "placental mesenchymal stem cells" refers to mesenchymal stem cells derived from the placenta. Thus in the present invention, and in particular in the context relating to the present invention, the term "placental mesenchymal stem cells" may be used interchangeably with "placental stem cells", "mesenchymal stem cells", unless otherwise specifically indicated.

In the present invention, the term "PBS buffer" or "PBS" refers to a phosphate buffer. The general formulation and formulation of the PBS used in the context of the present invention, as well as their general properties such as pH value or pH range, are well known to those skilled in the art and are typically commercially available pre-formulations (or powders), e.g. the PBS used in the field of the present invention is typically a commercial buffer at pH7.4(± 0.1), e.g. HyClone brand PBS buffer; in the present invention, the composition of PBS buffer solution in the classical application of the art includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate, and PBS used in the present invention has the same composition as that in the present invention, unless otherwise specified.

In the present invention, the term "placenta" refers to the placenta of a newborn, and in particular to the placenta within 4 hours of birth.

The mesenchymal stem cells are adult stem cells with self-replication and multidirectional differentiation potential, have the advantages of easy separation, culture and amplification, low immunogenicity and no expression of Major Histocompatibility Complex (MHC) of type II, can be used by variant, have strong migration and immunoregulation capabilities, promote tissue damage repair and regeneration by a paracrine mode, and are ideal seed cells for regenerative medicine.

Premature Ovarian Failure (POF), a disease characterized by amenorrhea, estrogen deficiency, and elevated gonadotropin levels in women before age 40. The incidence rate of the traditional Chinese medicine is 1-3% in general population, 4-8% in secondary amenorrhea women and 0.01% before 20 years old. In recent years, the incidence rate of premature ovarian failure in women of childbearing age tends to rise year by year and to be younger, but the mechanism of premature ovarian failure is still unclear, and genetic factors, autoimmune factors, medical operation wounds, environmental infections and the like are mainly induced. No effective treatment method can radically cure POF, and Mesenchymal Stem Cells (MSCs) belong to a class of pluripotent stem cells of mesoderm and have the advantages of strong proliferation capacity, multidirectional differentiation potential, immunoregulation function, light xenotransplantation rejection, low configuration requirement, easiness in separation and culture and the like. At present, there are mainly three typical sources of MSCs for treating premature ovarian failure, such as Umbilical Cord Mesenchymal Stem Cells (UCMSCs), bone marrow mesenchymal stem cells (BMSCs), and adipose stem cells (ADSCs). In recent years, the application of mesenchymal stem cells at home and abroad obtains a preliminary research result in the treatment of premature ovarian failure.

A study on the molecular mechanism of human placental mesenchymal stem cells for treating premature ovarian failure of mice is carried out by taking placenta of fetuses born by caesarean section at term, separating and purifying to obtain the placental mesenchymal stem cells, randomly dividing 20C 57 premature ovarian failure mice into 2 groups, injecting physiological saline into a control group, injecting placental mesenchymal stem cell suspension into an experimental group, continuously injecting 10d, and detecting the change of related genes of ZCchc11, Angpt1 and Cx 4 ovaries. The results show that the flow cytometry detects positive expression of CD29, CD44 and CD90, and negative expression of CD45 and HLA-DR. The fluorescent quantitative polymerase chain reaction detection result shows that compared with a control group, the expression of ZCchc11 and Angpt1 genes of the observation group is increased, the expression of Cxcr4 genes is reduced, and the difference has statistical significance (P < 0.05). The results show that the human placenta mesenchymal stem cells can regulate the expression of the ovary growth related genes and have an important effect on treating premature ovarian failure.

The method is characterized in that a model transplantation marker UCMSCs is established, and the distribution condition of green fluorescent cells of each organ is observed. The result shows that transplanted cells are found in the ovary, more transplanted cells can be seen in the lung and the spleen, and scattered transplanted cells can be seen in the heart, the liver and the kidney. These results indicate that UCMSCs can survive in ovarian tissues and partially reside in lung, spleen and other tissues.

A Zhushafang paper (Zhushafang, etc., PKH 26-labeled human umbilical cord mesenchymal stem cells in the ovary migration of a rat chemotherapeutic premature ovarian failure model, the development of modern obstetrics and gynecology, 2011 02) marks human umbilical cord mesenchymal stem cells with PKH26, discusses the ovary migration tracing of the human umbilical cord mesenchymal stem cells in the rat chemotherapeutic premature ovarian failure model, and the method comprises the steps of extracting and culturing the human umbilical cord mesenchymal stem cells, labeling the cells according to a PKH26 labeling program, observing the ultrastructure of labeled group cells and unlabeled group cells by a transmission electron microscope, and determining the proliferation, period and apoptosis conditions of the cells. And observing the ovary condition of the human umbilical cord mesenchymal stem cells marked by the PKH26 after transplantation under a fluorescence microscope. The results show that the morphology of the marked cells has no obvious difference, the marked cells are all fibroblast-like, the cell proliferation is not obviously influenced, the cell growth state is good, and the cells positive to the PKH26 mark are mainly in ovarian blood vessels. These results indicate that the PKH labeling technology can be used for the research of homing, differentiation and proliferation in the treatment of premature ovarian failure caused by chemotherapy in human umbilical cord mesenchymal stem cell transplantation.

The plum Yongli paper (plum Yongli, et al, experimental study on transplantation of human placental mesenchymal stem cells to repair premature ovarian failure of rats, world latest medical information abstracts, 2016 (90) period) aims at discussing the repair effect of tail vein injection transplanted human placental mesenchymal stem cells (hpMSCs) on premature ovarian failure model rat ovarian function caused by chemotherapy, the method comprises the steps of transplanting identified human placental mesenchymal stem cells to premature ovarian failure model rats through tail vein injection, dividing experiments into a hpMSCs group and a stem cell culture solution transplanting group (a control group), injecting 10 cells in each group respectively through tail vein isovolumic injection, observing the damage condition of rat ovarian structures after injection, and comparing the concentration of serum hormones (E2, AMH and INHB). The results show that the levels of the ovarian hormone of the rats are statistically different from the levels of the E2 and INHB in the serum before and after the transplantation of the hpMSCs compared with the control group; serum AMH levels were not statistically different (P ═ 0.051); the result of rat ovary histomorphology shows that the rat ovary cortex is seriously damaged compared with a model group, and the Bcl-2 expression is obviously reduced after tail vein injection of the hpMSCs. These results indicate that hAMSCs rat tail vein transplantation improves hormone levels and reduces ovarian cell apoptosis, thereby restoring ovarian function.

The fanxue paper (fanxue et al, the influence of mesenchymal stem cell transplantation on the ovary function of premature ovarian failure model mice, journal of old people in China, 2016 (21 st) discusses the influence of mesenchymal stem cells on premature ovarian failure model mice. 60 healthy ICR mice were randomly divided into control, model, and treatment groups. The control group mice do not intervene, and the model group mice and the treatment group mice are injected with cisplatin in the abdominal cavity to establish the animal model of the premature ovarian failure of the chemotherapy-induced damage mice. After the treatment model is built, the bone marrow mesenchymal stem cells are injected in situ for treatment. The general condition, the estrous cycle, of the rats were observed and the serum E2, FSH content and morphological changes of the ovarian tissues were determined. The results show that the estrus cycle of the mice in the model group is disordered, and the estrus cycle of the mice treated by the stem cells is basically recovered to be normal. Compared with the control group, the model group E2 decreased and the FSH level increased (P < 0.05). After the treatment of the bone marrow mesenchymal stem cells, E2 is increased, FSH level is reduced, and the normal level is nearly recovered. The model group showed a significant destruction of the follicles, with a significantly lower number of follicles than the control group (P < 0.01). The numbers of follicles and corpus luteum are obviously increased after the treatment of the bone marrow mesenchymal stem cells (including newly formed corpus luteum), and the difference is obvious compared with a model group (P < 0.01). These results show that cisplatin can cause apoptosis of follicular granule cells to cause premature failure of ovarian function, and mesenchymal stem cells can reduce damage of cisplatin to ovaries, promote follicular development, and improve and enhance ovarian function by regulating the content of each hormone in serum.

A waning Yan change paper (wang Yan, etc., research on the repair of ovary damage caused by VCD by mesenchymal stem cell transplantation, modern biomedicine progress, 10 2011) discusses the feasibility of mouse Mesenchymal Stem Cell (MSCs) transplantation on the treatment of premature ovarian failure caused by deoxyvinyl cyclohexene (VCD), and the method is to adopt VCD (160mg/kg/d) continuous intraperitoneal injection to induce the premature ovarian failure of mice. Injecting MSCs transfected with green fluorescent gene mouse bone marrow from each ovary, taking blood samples and ovary tissues of each group 14, 28 days and 45 days after transplantation, and observing the change of the estrus cycle of the mouse; detecting serum FSH and LH level by enzyme linked immunosorbent assay, and observing the distribution of MSC in ovary under microscope. The results show that after the MSCs are transplanted, green fluorescence can be seen in all groups, and the MSCs are mainly distributed in the ovarian interstitial region, and green fluorescence cells can also be seen in the ovarian cyst membrane cell region. The estrus cycle of the MSCs group is shortened compared with that of the experimental control group, the FSH and LH levels are lower than that of the experimental control group, and the difference is significant. These results indicate that bone marrow mesenchymal stem cells can improve ovarian endocrine function in premature ovarian failure mice and are present in ovarian tissues for a long time. The bone marrow mesenchymal stem cells can become a new method for treating premature ovarian failure.

The application value of the transplantation of the mesenchymal stem cells (BMSCs) in the reconstruction of the ovary function of the premature ovarian failure mice is discussed in a glittering and translucent paper (glittering and translucent paper, and the like, the transplantation of the mesenchymal stem cells is applied to the reconstruction of the ovary function of the premature ovarian failure mice, the school report of Anhui agriculture university, and the 01 th 2017). A mouse Premature Ovarian Failure (POF) model is established by one-time intraperitoneal injection of cyclophosphamide and busulfan, and is randomly divided into a building block group, a BMSCs transplanting group and a blank control group. Transplantation group in modeling 14d Green Fluorescent Protein (GFP) transgenic mouse BMSCs suspension (5X 10)⁷one/mL), repeated 1 time every 4d for 5 times; the building group and the blank control group are injected with physiological saline with the same amount. General conditions of the mice after modeling and after BMSCs transplantation, serum FSH and E2 levels, ovarian histological changes, and distribution of GFP fluorescence signals within the ovaries were observed. As a result, it was found that the appetite of the mice in the model group was significantly reduced, the activity was decreased, and the number of mice in the model group was reducedSignificant weight loss (P) compared to the white control group<0.05), FSH levels were significantly increased (P)<0.05), E2 level was significantly reduced (P)<0.05), ovarian volume decreased, interstitial fibrosis severe, and a significant decrease in primordial, growing and mature follicles (P)<0.05). After BMSCs are transplanted, the weight of transplanted mice is increased, the appetite is enhanced, and the activity is increased; FSH concentration after 28d was significantly lower than the set-up (P)<0.05), the E2 concentration, primordial follicle, number of growing follicles and number of mature follicles were all significantly higher than in the modeled group (P)<0.05), but each index still has significant difference with the blank control group; under the fluorescence microscope, green fluorescence signals can be seen and distributed around the follicles. The result indicates that the BMSCs can be positioned and survive in the damaged ovarian tissues of the mice and have the repairing effect on the ovarian tissue structures and the endocrine functions.

The aged sunset paper (aged sunset, et al, GDF-9 transfected adipose-derived mesenchymal stem cell for treating chemotherapeutic Premature Ovarian Failure (POF) rats, advanced in modern obstetrics and gynecology, 11 years 2017) studies the effect of the rat adipose-derived mesenchymal stem cell (ASC) which up-regulates the expression of growth differentiation factor-9 (GDF-9) for infusion treatment of chemotherapeutic Premature Ovarian Failure (POF) rats by culturing the ASC from rat fat by an enzymolysis adherence method, transfecting a gfp-N3-GDF-9 plasmid into the ASC by a liposome method, and verifying the expression of GDF-9. Injecting cisplatin into abdominal cavity to establish rat POF model, infusing ASC and ASC-GDF-9 cells via tail vein after 1 week, and observing rat weight, ovary weight, and estradiol (E) in serum₂) Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) values, and assayed for follicular formation. The results show that the ASC cells grow vigorously and can differentiate into osteoblasts and adipocytes, GDF-9 protein can be expressed smoothly after transfection, and the transfection efficiency is (43 +/-9.68)%. The cisplatin intraperitoneal injection can reduce the weight of a rat, reduce the ovary volume, reduce all levels of follicles and reduce E₂Decreased, apparent increase in FSH and LH. After 4 times of ASC-GDF-9 cell infusion, the number of follicles at all levels and the density of stromal cells can be obviously increased, and the hormone levels of LH and FSH are reduced. These results indicate that ASC transfected with GDF-9 can better improve ovarian function, restore development of all levels of follicles, and improve hormone disorder after tail vein infusion.

The Chenjing paper (Chenjing, et al, the role of mesenchymal stem cell transplantation in ovarian premature ovarian failure and reproductive function reconstruction in Anhui medical university, 11 years 2017) discusses the role of mesenchymal stem cell (BMSCs) transplantation in ovarian premature ovarian failure and reproductive function reconstruction, and the method comprises the steps of establishing an ovarian premature ovarian failure mouse model by one-time intraperitoneal injection of cyclophosphamide and busulfan, and dividing experimental animals into a building block group, a transplantation group and a blank control group. And 14d after modeling, injecting BMSCs of the Green Fluorescent Protein (GFP) transgenic mice into the mice of the transplant group through tail veins, and injecting an equal amount of physiological saline into the mice of the building group and the blank control group through the tail veins. And (3) detecting the levels of Follicle Stimulating Hormone (FSH) and estradiol (E2) in each group at 14d after modeling and 2 months after the BMSCs are transplanted, observing the positioning of the BMSCs in ovarian tissues and the apoptosis of ovarian granule cells after the BMSCs are transplanted for 2 months, and recording the number of eggs obtained, the fertilization rate, the cleavage rate and the blastocyst formation rate after the superovulation of each group, and the number of pregnancies and the fertility generations after the BMSCs are combined with a male mouse. The result shows that 2 months after the transplantation of the BMSCs, a large number of green fluorescent cells and marked BMSCs are visible in the ovarian stroma of the transplanted group, but FSH receptors are not expressed; the FSH level and the granulosa cell apoptosis index are obviously lower than those of a building module (P <0.05), the E2 level, the egg obtaining number, the fertilization rate, the cleavage rate and the blastocyst formation rate are all obviously higher than those of the building module (P <0.05), but all indexes are still obviously lower than those of a blank control group (P < 0.05); the model building group has no mouse pregnancy, 2 mice in the transplantation group are pregnant, and 4 and 2 mice with normal appearance are respectively bred. These results indicate that BMSCs can localize in ovarian stroma, significantly reduce ovarian granule apoptosis index, improve ovarian endocrine function, and improve mouse fertility.

The treatment effect of heat shock pretreated Mesenchymal Stem Cells (MSCs) on cyclophosphamide-damaged rat ovary was discussed in a Wanglong (Wanglong, etc., effect of heat shock pretreated mesenchymal stem cell transplantation on chemotherapy premature ovarian failure, Guangdong medicine, 2018, stage 13) by isolating and culturing rat bone marrow MSCs and pretreating at 42 ℃ for 1h before transplantation. The experiments were divided into 4 groups: normal, model, MSCs, heat shock groups, 25 rats per group. The normal group is not treated, and the other 3 groups establish chemotherapy-induced premature ovarian failure animal modelsType, MSCs group rat bilateral intra-ovarian injection of 1 × 10⁶Rats with MSCs in heat shock group injected with 1 × 10 bilaterally in ovaries⁶Heat shock pretreated MSCs. The vaginal smear monitors the estrous cycle of the rat, and the chemiluminescence method detects estradiol (E2), and the radioimmunoassay detects Follicle Stimulating Hormone (FSH). Blood was collected from each group on days 1, 15, 30, 45, and 60 after modeling and 5 rats were sacrificed from each group to observe ovarian weight, ovarian structure, and number of follicles at each level. The results showed that the heat shock group rats restored the estrous cycle faster than the MSCs group; the ovarian weight, total number of follicles and number of follicles at each stage of the heat shock group and the MSCs group were statistically different from those of the model group (P)<0.05), the difference between the heat shock group and the MSCs group was also statistically significant (P)<0.05); at 30, 45 and 60 days after transplantation, the difference between the concentration of MSCs, E2 and FSH in the heat shock group and the model group is statistically significant (P)<0.05), the comparative difference between the heat shock group and the MSCs group was also statistically significant (P)<0.05). These results indicate that heat shock pretreated MSCs have a stronger reparative effect on chemotherapeutic premature ovarian failure.

Zhanglingli paper (Zlingli, et al, experimental study on feasibility and mechanism of POF rat treatment by human umbilical cord mesenchymal stem cells, Chongqing medicine, 31 years 2018) discusses feasibility and possible mechanism of POF rat treatment by human umbilical cord mesenchymal stem cells (hUCMSCs), the method comprises dividing 62 healthy SD rats into a control group (group A), a model group (group B) and a hUCMSCs transplantation group (group C), injecting cyclophosphamide into abdominal cavities of B, C groups of rats to establish POF animal models of chemotherapy-induced rats, injecting isovolumetric sterilized water into the group A of rats, and after successful modeling, injecting hUCMSCs into the group C of rats through tail vein to treat the group C. The serum levels of estradiol (E2), Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), inhibin B (INHB), anti-Mullerian hormone (AMH), cysteine-containing aspartate proteolytic enzyme 3(caspase-3), and the distribution of the human ALU gene in the ovaries of rats were compared among the groups of rats, and the morphology of the ovarian tissues, the number of follicles, and the number of corpus luteum after HE staining of the rats were compared among the groups of rats. The results show that 1d after modeling is completed, the INHB and AMH levels of the group B are lower than those of the group A; after stem cell transplantation, levels of INHB and AMH in group C are increased, ovary volume is increased, growing follicles are increased, and closure is stoppedThe locked follicles were reduced and the difference compared with group B was statistically significant (P)<0.05). Human ALU gene and caspase-3 signal expression level [ (4.40 +/-1.46) x 10 ] are observed in ovarian stroma, ovarian granules and ovarian daughter cells^-3]Significantly lower than in group B (P)<0.05). These results indicate that hUCMSCs act on ovaries through two ways of repairing granulosa cells and directly synthesizing granulosa cells into ovum cells, so that the POF type rats can be treated.

Dujing paper (Dujing, et al, the influence of human umbilical cord mesenchymal stem cells on ovarian ultrastructure and function of premature ovarian failure rats, advanced in modern obstetrics and gynecology, 2018, stage 02) discusses the influence of human umbilical cord mesenchymal stem cells (hUCMSCs) on ovarian ultrastructure and storage function of premature ovarian failure model rats, by randomly dividing 62 healthy SD rats into a negative control group (18 cases), a model control group (22 cases) and a hUCMSCs transplantation group (22 cases). The rats in the negative control group are not treated, and the rats in the model control group and the hUCMSCs transplantation group are injected with Cyclophosphamide (CTX) in the abdominal cavity to establish an ovarian premature senility animal model. After successful modeling, the transplant group of hUCMSCs was treated by tail vein injection of hUCMSCs. Observing general condition of rat, observing change of ultrastructure of rat ovary granular cell, and measuring estradiol (E) in serum₂) Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), inhibin b (inhb), and anti-mullerian hormone (AMH) levels, and analyzing changes in ovarian reserve function. The results show that under an electron microscope, the number of mitochondria and endoplasmic reticulum is gradually reduced due to karyolysis, nuclear membrane disappearance and mitochondria vacuolation of the model control group particles; the nucleus membranes of granular cells of the hUCMSCs transplanted group are gradually repaired, nucleoli appears, and endoplasmic reticulum and mitochondria are slightly existed. 1d after model building, compared with a negative control group, the E2 content of the model control group and the hUCMSCs transplanting group is reduced, the FSH content is increased, and the difference has statistical significance (P)<0.05); 1d after the transplantation of the hUCMSCs, the INHB and AMH contents of the model control group and the hUCMSCs transplantation group are lower than those of the negative control group, and the difference has statistical significance (P)<0.05); 17d after the transplantation of the hUCMSCs, the INHB level of the hUCMSCs transplantation group is higher than that of the model control group, and the difference has statistical significance (P)<0.05). These results indicate that hUCMSCs transplantation has certain repair effect on the ovarian ultrastructure and storage function of POF ratsThe application is as follows.

A paper of beauty (beauty, et al, J.Zhonghua Progenity and contraception, 2018, stage 02) discusses the effect of human placental mesenchymal stem cell (hPMSC) transplantation therapy on the expression of cyclophosphamide-induced Premature Ovarian Failure (POF) model SD rat superoxide dismutase 1(SOD1) and uncoupling protein-2 (UCP-2). The method is to randomly divide 60 female SD rats into 4 groups (n is 15): blank control group, POF model group, treatment control group, hPMSC transplant group. Injecting cyclophosphamide solution into abdominal cavity of rat with first dose of 50mg/kg and maintenance dose of 8mg/kg for 14 days to establish POF model, observing estrus cycle change of rat, and determining serum estradiol (E2), Follicle Stimulating Hormone (FSH), anti-mullerian hormone (AMH), Reactive Oxygen Species (ROS), 8-hydroxydeoxyguanosine (8-OHdG) levels by ELISA method; detecting the morphology and follicle count of the ovarian tissue by an HE staining method, and observing the fibrosis condition of the ovarian tissue by a VG staining method; SOD1 and UCP-2 protein expression were determined by immunohistochemistry and Western blotting. The results show that the ELISA results show: the serum FSH level [ (10.60 +/-1.0) IU/L ] of the model group is obviously higher than that of the blank control group [ (5.83 +/-0.92) IU/L ] (P <0.01), while the E2 and AMH levels [ (35.52 +/-10.27) ng/L and (2090.6 +/-397.5) ng/L ] are obviously lower than that of the blank control group [ (65.62 +/-3.76) ng/L and (3636.39 +/-204.46) ng/L ], and the difference has statistical significance (P < 0.01). The hPMSC transplant group had elevated ROS and 8-OHdG in serum compared to the blank control group (P < 0.05). The follicle count showed that the number of atretic follicles in the hMSC-transplanted group (5.36 ± 1.11) was significantly lower than that in the treated control group (8.01 ± 2.22) and the POF model group (11.21 ± 1.69) (P < 0.05). ROS and 8-OHdG were significantly reduced in the hPMSC transplanted group compared to the treatment control group (P < 0.05). VG staining showed a significant decrease in the fibrotic area ratio of the hPMSC-transplanted group compared to the treatment control group (P < 0.05). Western blotting results show that compared with a treatment control group, the expression levels of SOD1 and UCP-2 proteins in the hPMSC transplant group are remarkably reduced (P <0.05), and the difference between the hPMSC transplant group and the control group has no statistical significance (P > 0.05); immunohistochemical results showed that the expression of SOD1 and UCP-2 proteins was reduced in the model group compared to the treatment control group, whereas the hPMSC transplantation group was statistically insignificant compared to the blank control group. These results indicate that hPMSC transplantation improves the oxidative stress of POF model SD rats, reduces SOD1 and UCP-2 expression, and plays a role in protecting the function of ovarian mitochondria.

The inventor of the invention utilizes a perfusion method to separate and culture the mesenchymal stem cells from the placenta, and obtains the mesenchymal stem cells with high purity. However, after perfusion, a large amount of stem cells still remain in the placenta tissue and cannot be effectively separated. Therefore, it is considered that the mesenchymal stem cells cannot be obtained to the maximum extent by the perfusion method.

The invention discloses a method for separating a large number of mesenchymal stem cells from placenta, and the method is used for preserving the mesenchymal stem cells of the placenta and establishing a placenta stem cell bank. Based on the summary of the conventional separation and culture of the mesenchymal stem cells, the inventor successfully separates and obtains a large amount of mesenchymal stem cells from the placenta by utilizing a mixed digestion of a plurality of tissue digestive enzymes and combining an adherence culture method. The mesenchymal stem cells obtained by the method have high purity and large quantity, have the same biological characteristics as the mesenchymal stem cells of the bone marrow, and can be differentiated into osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells and the like. Because stem cells in the placenta are more immature than adult stem cells and rich in content, the placenta stem cells have wide application prospects in clinic, the mesenchymal stem cells are cryopreserved like cord blood by using a conventional cell cryopreservation method, a placenta stem cell bank is established, and a foundation is laid for the further research and clinical treatment of the stem cells.

Because the cord blood contains abundant hematopoietic stem cells, people establish a cord blood bank to store the cord blood hematopoietic stem cells which are an important biological resource, and a treatment means is provided for various blood system diseases and immune system diseases. Similarly, the placenta mesenchymal stem cells are used as a more important stem cell resource, and are frozen in liquid nitrogen at the deep low temperature of-196 ℃ for long-term storage by using a conventional cell freezing storage method, so that a placenta stem cell bank is established, and seeds are preserved for the treatment of stem cells in the future.

Particularly, the method can obtain primary cells with extremely high mesenchymal stem cell purity in the P0 generation, the expression of CD73 of the primary cells is more than 60 percent, the expression of CD45 of the primary cells is not carried out, and the content of the mesenchymal stem cells in the primary cells reaches 60 to 70 percent.

The technical effect of the method of the invention is obvious. For example, the invention selects a full-term placenta sample, cuts 15g of tissue at a specific position of a placenta lobule, digests the tissue by using a mixed enzyme system, obtains cells, and then purifies the cells to obtain a group of relatively pure mesenchymal stem cells (the expression of CD73 is more than 60 percent, and the expression of CD45 is not carried out), wherein the number of the obtained cells per gram of the tissue can reach 2.5 multiplied by 10⁷And the yield is stable, and the sample specificity is greatly reduced. The primary mesenchymal stem cells are subjected to recovery culture after cryopreservation, and are cultured by using a classical complete culture medium formula, so that more spindle-type adherent cells can be microscopically detected in about 4 days, the cell fusion rate reaches 70-80% in 10 days, and the cells can be transferred to P1 generations. After continuous passage to P5 generation, carrying out experiments such as flow type phenotype identification, cell cycle detection, induced differentiation and the like, wherein the results show that the cells of P1-P5 generation are mesenchymal stem cells, the positive expression (CD73, CD90 and CD105) is more than 98 percent, and the negative expression (CD34, CD45, CD19, CD11b and HLA-DR) is less than 2 percent; the G2 stage cells of the P5 generation cells are less than 1 percent, have strong proliferation capacity and do not enter the division stage; the ability to differentiate into osteoblasts, adipoblasts and chondroblasts under the stimulation of a specific induction medium.

The method is simple to operate, convenient and practical, can obtain a large amount of mesenchymal stem cells, has good differentiation performance, and has the capacity of differentiating osteoblasts, adipocytes, chondrocytes, endothelial cells, nerve cells and other cells. Comparison with existing methods: at present, MSC is mainly obtained by extracting donor bone marrow by an operation method or separating placenta by a perfusion method and carrying out adherent culture. The method has the advantages of low cell number, and infection possibility in marrow extraction and after marrow extraction. The invention successfully separates and obtains a large amount of mesenchymal stem cells with higher purity from the placenta, and establishes a placental stem cell bank by using the method to store the stem cells with great application prospect. The method is simple and easy to implement, and because the placenta is the same as cord blood, the cell components are more immature, the source is wide, and the method is convenient and easy to obtain, the method has wide prospect in the clinical application of stem cells.

Drawings

FIG. 1: the flow type phenotype identification result of the primary cell obtained by the invention is shown in the figure.

FIG. 2: micrographs of samples during passage P0.

FIG. 3: micrographs of samples during passage P5.

FIG. 4: the samples were phenotyped at P5.

FIG. 5: DNA content-cell number relationship of the sample P5 generation cells.

FIG. 6: the induced differentiation test of the P5 generation cells shows that the cells have the capacity of differentiating to osteogenic, adipogenic and chondrogenic cells.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

Example 1, placental whole cell treatment:

1. preparing a mixed synthase digestive solution: 22ml of HBSS (Hank' S balanced salt solution) containing calcium and magnesium ions, 0.4ml of Liberase Roche MNP-S enzyme (purchased from Sibao, for example, cat # 5578582001) and 0.7ml of DNA I enzyme were transferred into a 50ml centrifuge tube, and zinc chloride (added at a concentration of 0.2g/L, 0.25g/L or 0.3g/L) was added thereto, mixed, and preheated at 37 ℃ for 20min or more. The Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl2 & 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4.

2. Preparation of placental leaflets: the placenta is taken out from the collection bag and placed in a white porcelain dish, the blood stasis of the placenta is removed after the tissue cleaning solution is washed, and a small amount of placenta lobule tissue (about 20 g) is cut and placed in a steel cup. The tissue is washed twice with tissue washing solution (0.9% physiological saline + double antibody (double antibody is streptomycin, content 1%)), and after soaking for 5min, 15g + -1 g of better tissue is weighed into a 100mm glass dish.

3. Removal of blood cells: adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm³About the size, 100ml of tissue cleaning fluid is added and stirred evenly, then the mixture is filtered by a 300-mesh filter screen, and then the mixture is cleaned twice by the tissue cleaning fluid (the leaflet tissue is moved into a steel cup each time, 100ml of tissue cleaning fluid is added and stirred evenly, and then the mixture is filtered by a 300-mesh filter screen).

4. Mixed enzyme digestion and termination: adding the cleaned leaflet tissue into preheated 23ml of mixed enzyme digestive juice, fully and uniformly mixing, and then oscillating and digesting for 30min by a shaking table at 37 ℃ and 100 rpm. After digestion, the tissue fluid +2ml FBS was terminated.

5. Collecting primary cells:

adding 50ml of tissue cleaning fluid to dilute and mix the tissue fluid uniformly, filtering by a 300-mesh sieve, collecting cell fluid, washing the digested tissue twice (50 ml of tissue cleaning fluid is used each time), merging the filtrate into 1 250ml of centrifuge tubes, and centrifuging at 1500rpm for 8min (acceleration 9 and deceleration 7);

removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, centrifuging at 1500rpm for 8min (acceleration 9, deceleration 7);

removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells; 1ml of suspension is taken and used for a sysmex hematology analyzer to count cells, and the primary cells are high in purity and have the mesenchymal stem cell content of about 60-70 percent through determination.

6. Freezing and storing primary cells:

the frozen stock solution is prepared on site, and the formula of the frozen stock solution is as follows: 65% DMEM-F12, 15% Human Serum Albumin (HSA), 20% DMSO, e.g., WAK brand DMSO, ready for use;

centrifuging the cell suspension at 1800rpm for 10min (acceleration 9 and deceleration 7), collecting cell precipitate and supernatant 5ml (sample of supernatant 10 ml), re-suspending, slowly adding the prepared frozen stock solution while shaking;

the cell suspension was dispensed into 92 ml cryopreserved tubes, 1.5ml per tube (pre-cooled in a programmed cooling box). The remaining cell suspension + the retained sample supernatant are used for sterile detection;

and (4) performing program cooling by using a program cooling instrument, transferring the cells into a liquid nitrogen storage tank, and performing cryopreservation on the obtained primary cells.

Through the whole placenta cell processing process of the embodiment 1, a placenta sample of full term is selected, 15g of tissue at a specific position of a placenta lobule is cut, the placenta sample is digested by the mixed enzyme digestive fluid system, the cells are obtained and then purified, and a group of relatively pure primary mesenchymal stem cells (the expression of CD73 is more than 60 percent, and the expression of CD45 is not carried out) can be obtained, the content of the mesenchymal stem cells in the primary mesenchymal stem cells reaches 60 to 70 percent, and the number of the primary cells obtained by each gram of the placenta lobule tissue can reach (2.4 to 2.8) multiplied by 10⁷Moreover, the yield is stable, and the sample specificity is greatly reduced. However, when the mixed enzyme digestive fluid is not added with the zinc chloride, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually in the range of 31-38 percent, and the number of the primary cells obtained per gram of the placenta lobule tissue is less than 5 multiplied by 10⁵A plurality of; in addition, the present inventors found that the number of primary cells obtained per gram of placental lobule tissue was less than 2X 10 when the primary cell preparation was performed with reference to other prior art techniques⁶And 1/10 or less in the method of the present invention. According to the invention, the whole cell treatment of the placenta can efficiently obtain primary mesenchymal stem cells, and a good foundation is laid for the subsequent culture of the mesenchymal stem cells with high medical value.

For example, in this example, the cell yield was very stable after placental tissue treatment, and typical data for some experiments are shown in table 1.

Table 1: cell yield of primary cells from placental tissue

Date of treatment	Sample registration number	Tissue volume (g)	Number of cells (. times.10)⁸)	Cell yield (10)⁸/g)
					2016.7.22	9004116082279	15	3.8	0.25
2016.7.25	9004116082301	15.9	3.9	0.25
					2016.7.26	9004116082311	14.9	3.8	0.26
2016.7.27	9004116082319	14.9	3.8	0.26
					2016.8.25	9004116082539	15	3.75	0.25

In addition, the flow type phenotype identification result of the primary cells obtained after the placenta treatment shows that the expression of CD73 is up to more than 60%, and CD45 is not expressed, which indicates that the primary cells are a group of relatively pure mesenchymal stem cells and do not contain blood cells. The flow phenotyping results are shown in FIG. 1.

Example 2 recovery and subculture of Primary cells

1. Cell recovery:

taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; as not otherwise specified, complete medium as used herein is DMEM-F12 medium containing 10% FBS;

centrifuging at 1200rpm for 5min (acceleration 9, deceleration 7), removing supernatant, and adding 5ml complete culture medium for resuspension; inoculating each tube of cells into 1T 75 culture bottle, supplementing complete culture medium to 30ml, and culturing in CO2 incubator (37 deg.C, 5% CO2, saturated humidity); performing total liquid change every 3-4 days with complete culture medium, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm²The following passages can be performed;

2. cell passage: washing recovered P0 generation cells with PBS, adding 2ml of pancreatin for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9, deceleration 7), discarding supernatant, adding 5ml of complete culture medium for resuspension, counting, and inoculating to a culture bottle, wherein the cell density is 8000-12000 cells/cm²Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation;

the passage operations from the generation P0 to the generation P1 are sequentially repeated to perform cell passages from the generation P1 to the generation P2, from the generation P2 to the generation P3, from the generation P3 to the generation P4 and from the generation P4 to the generation P5 respectively, so that the mesenchymal stem cells of each generation are obtained.

In this example, the seeding density was about 5X 10⁵cells/cm²And a plurality of adherent cells are arranged in the field of microscopic examination for 4 days after inoculation and are spindle-shaped. The strain can be transferred to P1 generation 10 days after inoculation. The cells grow fast and are full, and the cells are in spindle shapes. During passage from P0 to P1, the cell counts for some experiments are shown schematically in table 2 below. The micrograph of the PS162279 sample during passaging is shown in FIG. 2.

Table 2: cell count results during passage from passage P0 to passage P1

Sample registration number	P0-P1	P0 count
			PS162311	D10	8*10⁵(ADAM)
PS162319	D10	1.4*10⁶(ADAM)
			PS162279	D9	1.5*10⁶(ADAM)

In addition, in the process of inoculating and passaging P1-P5, the seeds are usually cultured for 4-5 days, harvested and passaged to the next generation. An exemplary micrograph of the PS162279 sample during passage P5 is shown in figure 3.

In this experiment, the flow phenotype identification of P1-P5 generation was carried out, the results showed that the positive expression of CD73, CD90 and CD105 was > 98%, and CD34, CD45, CD19 and HLA-DR were identified, and the results are shown in Table 3, which proved that the cells isolated and cultured in placenta were mesenchymal stem cells and had high purity.

Table 3: flow phenotyping results of P1-P5 generation cells

Exemplary, the P5 representative flow phenotypic identification results of the PS162279 sample are shown in fig. 4.

In addition, the growth cycle of some samples was measured for P5 generation cells, and the results showed < 1% G2 phase cells and > 10% S phase cells, demonstrating that these cells are highly proliferative and do not enter the division phase, and the specific results are shown in Table 4.

Table 4: growth cycle assay results for P5 generation cells

Sample registration number	Stage GO/G1	Stage S	Stage G2/M
				PS162311-P5	84.60％	14.80％	0.64％
PS162319-P5	82.30％	16.80％	0.93％
				PS162279-P5	87.00％	11.90％	0.80％

In addition, the DNA content-cell number relationship was plotted for PS162311-P5 cells, and typical results are shown in FIG. 5.

Example 3 biological characterization of placental MSCs

The biological characteristics of the placenta mesenchymal stem cells are identified by referring to the methods of issued patents CN102676451A [0062] to [0089], and the results show that the MSC separated by the method has the capacity of differentiating into osteoblasts, adipocytes and chondrocytes, thereby confirming that the MSC obtained by the method has the characteristics of stem cells.

For example, the P5 generation cells were illustratively tested for induced differentiation and showed the ability to differentiate into osteogenic, adipogenic and chondrogenic cells. A typical micrograph of adipogenic, osteogenic, and chondrogenic differentiation is shown in fig. 6.

Example 4 effectiveness of mesenchymal Stem cells in treating POF

(1) 6-week-old female C57BL/6 mice were intraperitoneally injected with 50 mg/kg/day Cyclophosphamide (CTX) for 15 days at the same time every day to establish a POF mouse model. Ovarian reserve function is assessed by indicators such as follicle number, hormone levels, oestrus cycle and fertility tests.

(2) Female C57BL/6 mice, 6 weeks old, were randomized into: control group (n ═ 20), POF model group (n ═ 20), placental mesenchymal stem cell treatment group a (PD-MSC group a, n ═ 20), placental mesenchymal stem cell treatment group b (PD-MSC group b, n ═ 20). Treatment groups of mice, 2 injections of placental mesenchymal stem cell preparation, 2 × 10 per mouse, were administered on days 1 and 14 after POF modeling, respectively⁵Tail vein injection of placenta mesenchymal stem cells. The POF model group was injected with an equal volume of saline. In the present experiment, the placental mesenchymal stem cell injection preparation was prepared as follows: transferring the mesenchymal stem cells (in this case, PS162279 sample P5 generation) obtained by the cell passage of step 2 of example 2 into a centrifuge tube, centrifuging at 1500rpm for 5min, discarding the supernatant, adding 0.9% sodium chloride solution, and resuspending to obtain the cells with concentration of 1-3 × 10⁶A cell preparation of individual placental mesenchymal stem cells/ml; wherein, the 0.9% sodium chloride solution is additionally added with magnesium gluconate and phospholipid (magnesium ion concentration is 5mmol/L, phospholipid concentration is 0.2mg/ml, phospholipid is from injection grade soybean, and the group added with the magnesium gluconate and the phospholipid is marked as PD-MSC group a) or not added with the magnesium gluconate and the phospholipid (the group added with the magnesium gluconate and the phospholipid is marked as PD-MSC group b).

(3) Hormone levels

After the placenta mesenchymal stem cells are transplanted for the second time for 14 days and 28 days, 10 mice are respectively taken from each group, blood is collected from the orbit, serum is separated, and the mixture is preserved at the temperature of minus 20 ℃. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze the levels of estradiol (E2), Follitropin (FSH), and anti-Mullerian hormone (AMH) (the specific method was performed according to the method of the corresponding publication). The results show that: compared with the POF model group, the level of E2 in the serum of mice in the placenta mesenchymal stem cell group is increased at each time point, the level of FSH is reduced, and the difference is obvious (p is less than 0.05), and the specific results are shown in the following table.

P <0.05, p <0.01, compared to the POF model groups on the same assay day.

(4) Follicle count of mouse ovarian tissue

28 days after the placenta mesenchymal stem cells are transplanted for the second time, 10 mice are taken from each group and killed, the left ovary tissue of each mouse is taken and fixed in 4 percent paraformaldehyde, the fixed tissue is dehydrated by series alcohol, xylene is transparent, paraffin is embedded, the serial sections are cut, the section thickness is 5um, HE staining is carried out, and observation is carried out under a microscope.

The results show that: compared with a control group, the number of primary follicles, secondary follicles and mature follicles of the mice in the POF model group is obviously reduced, and the number of atretic follicles is obviously increased; after 28 days of treatment of the placenta mesenchymal stem cells, the number of all levels of follicles is recovered to different degrees, the growth of granulosa cells is increased, apoptosis is reduced, the form of ovarian epithelial cells is stable, the number of primary follicles, secondary follicles and mature follicles is obviously increased, and the number of atretic follicles is obviously reduced. The counting of each stage of follicles in 28 days after the placenta mesenchymal stem cell transplantation is obviously different from that of the POF group, P is less than 0.01, and specific results are shown in the following table.

	Control group	POF model set	PD-MSC group a	PD-MSC group b
					Primary follicle	26.64±3.26	7.12±1.94	21.17±4.15**	10.64±1.93
Secondary follicle	24.87±4.34	7.41±2.82	22.42±3.87**	13.26±2.24*
					Mature follicle	23.92±2.62	6.86±2.11	20.15±4.03**	9.83±1.74
Atresia follicle	2.64±1.04	6.03±1.36	2.87±0.92**	5.26±1.16

P <0.05, p <0.01 compared to the POF model group.

(5) Observation of mouse fertility:

on day 28 after placental mesenchymal stem cell transplantation, male and female mice were transplanted as 2: 1 proportion is bred in a cage, the fertility rate of the mice is counted, the litter size of the mice is compared, the repairing effect of placenta mesenchymal stem cell transplantation on the ovary function of the mice is observed, the placenta mesenchymal stem cell group and the POF group have obvious difference, and P is less than 0.01. Specific results are shown in the following table.

	Control group	POF model set	PD-MSC group a	PD-MSC group b
					Number born	14.3±1.8	4.6±1.3	9.7±1.5**	6.4±1.9

P <0.05, p <0.01 compared to the POF model group.

According to the results, the placenta mesenchymal stem cell transplantation treatment can obviously improve the reserve function of the damaged ovary of the POF mouse, increase the number of follicles, increase the estrogen and the progestogen, recover the fertility of part of mice, and provide experimental basis for applying the placenta mesenchymal stem cell to the clinical treatment of the POF.

Example 5 establishment of placental stem cell Bank

1. Detection of cellular Activity

The number of viable cells before and after cryopreservation was counted using trypan blue staining.

2. Detection of cell contamination

And (3) detecting whether the cells are polluted by fungi and bacteria by using a small amount of cell culture. And (3) detecting whether the cells are infected by hepatitis B two-and-two, hepatitis C, AIDS, cytomegalovirus, EB virus, syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM, EBV-IgA and TRUST by utilizing an etiological method.

3. Detection of genetic disorders

And detecting whether the frozen cells have genetic diseases or not by using a molecular genetics method.

4. HLA-ABC/DR match

Cells were tested for HLA-ABC/DR phenotype and recorded.

5. Investigation of cell origin

The details of the fetus and its parents are recorded and recorded on the record.

6. Establishment of placental stem cell database

After normal placental stem cells are preserved, a database of placental stem cells is established, which includes the first six data, and associations with cryopreserved cells are established.

Claims

1. A cell preparation is a cell suspension prepared by suspending placenta mesenchymal stem cells in 0.9% sodium chloride solution, wherein the cell concentration is 1-10 multiplied by 10⁶Magnesium gluconate and phospholipid are also added into the 0.9 percent sodium chloride solution, wherein the magnesium gluconate is added in an amount that the concentration of magnesium ions is 5mmol/L and the concentration of phospholipid is 0.2 mg/ml; the cell preparation is prepared by a method comprising the following steps: transferring the mesenchymal stem cells obtained by cell passage to a centrifuge tube, centrifuging, discarding supernatant, adding 0.9% sodium chloride solution containing magnesium gluconate and phospholipid, and resuspending to obtain cell preparation; the mesenchymal stem cell is prepared by a method comprising the following steps:

(1) treatment of placental leaflets: placing placenta in a white porcelain dish, washing with tissue cleaning solution to remove blood stasis of placenta, shearing 20g of placenta lobule tissue in a steel cup, cleaning twice with the tissue cleaning solution, soaking for 5min, and weighing 15g of better tissue in a 100mm glass dish; adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm³About the size, 100ml of tissue cleaning fluid is added, the mixture is stirred evenly and filtered by a 300-mesh filter screen, and the operation is repeated to clean the tissue cleaning fluid twice so as to remove blood cells; the tissue washing solution is 0.9% physiological saline containing 1% double antibody;

(2) mixed enzyme digestion and termination: adding the cleaned leaflet tissue into 15-30 ml of mixed enzyme digestive juice preheated at 37 ℃, fully mixing uniformly, then digesting for 30min by shaking at 37 ℃ and 100rpm in a shaking table, and after digestion is finished, adding 2ml of FBS into the tissue juice to stop digestion;

(3) collecting primary cells: adding 50ml of tissue cleaning fluid into the tissue fluid obtained in the last step, uniformly mixing, filtering by a 300-mesh sieve, and collecting cell fluid; washing the digested tissue twice repeatedly, combining the filtrates of the two times into a centrifuge tube, and centrifuging at 1500rpm for 8 min; removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, and centrifuging at 1500rpm for 8 min; removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells;

(4) freezing and storing primary cells: centrifuging the cell suspension at 1800rpm for 10min, collecting cell precipitate and lower solution 5ml, re-suspending, slowly adding frozen solution 10ml while shaking; subpackaging the obtained cell suspension into 9 freezing tubes of 2ml, each tube of 1.5ml, placing in a precooled program cooling box, performing program cooling by using a program cooling instrument, and transferring the cells into a liquid nitrogen storage tank for freezing;

(5) cell recovery: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; centrifuging at 1200rpm for 5min, removing supernatant, and adding 5ml complete culture medium for resuspension; each tube of cells was inoculated into 1T 75 flask, supplemented with complete medium to 30ml, CO was added₂The temperature in the incubator is 37 ℃ and 5% CO₂Culturing under the condition of saturated humidity; performing total liquid change every 3-4 days with complete culture medium, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm²The following passages can be performed; the complete medium was DMEM-F12 medium containing 10% FBS;

(6) cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of cells fall off, adding 5ml complete culture medium to stop digestion, transferring cells into centrifuge tube, centrifuging at 1400rpm for 5min, discarding supernatant, adding 5ml complete culture mediumCounting after suspending and inoculating to a culture bottle, wherein the cell density is 8000-12000 cells/cm²Placing CO in₂The incubator is at 37 ℃ and 5% CO₂Culturing under the condition of saturated humidity until the cell density reaches more than 90 percent, and completing cell passage from P0 generation to P1 generation; repeating the operations in sequence to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain the mesenchymal stem cells of each generation.

2. The cell preparation according to claim 1, wherein the cell concentration is 1 to 5X 10⁶Individual cells/ml.

3. The cell preparation according to claim 1, wherein the cell concentration is 1 to 3X 10⁶Individual cells/ml.

4. The cell preparation according to claim 1, wherein in step (2), the mixed enzyme digest comprises: 15-30 volumes of Hank' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme and 0.2-2 volumes of DNA I-type enzyme.

5. The cell preparation according to claim 1, wherein in step (4), the formulation of the cryopreservation solution is: 65% DMEM-F12, 15% human serum albumin, 20% DMSO.

6. The cell preparation according to claim 1, further comprising, in the preparation of mesenchymal stem cells:

7. The cell preparation according to claim 6, further comprising, in the preparation of mesenchymal stem cells:

(8) freezing and storing the passage-generated placenta mesenchymal stem cells obtained in the step (6) in liquid nitrogen; and

(9) establishing a database of placental stem cells comprising the information detected in step (7) above, and correlating the database with the cryopreserved cells of step (8).

8. The cell preparation according to claim 1, wherein the resulting cell purity of each generation of placental mesenchymal stem cells is greater than 90%.

9. The cell preparation of claim 1, wherein said placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.

10. The cell preparation according to claim 4 wherein said Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO₄·7H₂O, 0.1g/L MgCl₂·6H₂O, 0.06g/L Na₂HPO₄·2H₂O, 0.06g/L KH₂PO₄1.0g/L glucose, 0.14g/L CaCl₂0.35g/L NaHCO₃0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4.

11. The cell preparation according to claim 4, wherein the mixed enzyme digest further comprises 0.2-0.3 g/L zinc chloride in addition to Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA type I enzyme.

12. Use of a cell preparation according to any one of claims 1 to 11 in the manufacture of a medicament for the treatment and/or prevention of premature ovarian failure.