CN109628388B - Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition - Google Patents
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Abstract
The present invention relates to the isolation of mesenchymal stem cells from placental blood vessels using a digestive enzyme composition. In particular to a digestive enzyme composition used in a method for separating placenta mesenchymal stem cells from placenta blood vessels, wherein a buffer solution containing tissue digestive enzyme is prepared by adding the following digestive enzyme into the buffer solution: pancreatin, deoxyribonuclease I, collagenase II, collagenase IV and hyaluronidase. In addition, the present invention relates to a method for isolating mesenchymal stem cells from placental blood vessels, comprising the steps of: disinfecting the placenta; stripping placental blood vessels from the placenta; cutting, cleaning, and filtering to remove blood stain to obtain placenta vascular tissue; adding mixed enzyme solution for digestion; stopping digestion, filtering and collecting tissue fluid; centrifuging the obtained cell sediment to obtain original placenta mesenchymal stem cells, re-suspending, sampling and counting the number and the survival rate of nucleated cells; freezing and storing the obtained cells, or continuously carrying out passage and/or carrying out cell identification, detection, freezing and storage, cell bank establishment and the like. The method can effectively improve the efficiency of separating the mesenchymal stem cells from the placental blood vessels.
Description
Technical Field
The present invention relates to a method for isolating stem cells from blood vessels of the placenta, and more particularly, to a method for isolating mesenchymal stem cells from blood vessels of the placenta using a digestive enzyme composition of the unique formulation of the present invention. The method can effectively improve the efficiency of separating the mesenchymal stem cells from the blood vessels of the placenta.
Background
Mesenchymal Stem Cells (MSCs), such as human mesenchymal stem cells, were first isolated from bone marrow, a class of tissue stem cells derived from the mesoderm that have multipotent differentiation potential and self-renewal capacity to differentiate into various adult cells, such as osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, hepatocytes, and the like, under specific conditions in vivo and in vitro (Caplan ai. Mesenchymal stem cells j ortho res.1991, 9. The latest research shows that the mesenchymal stem cells have the functions of immunoregulation and hematopoiesis 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 mainly come 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 taking process; because the content of MSC in human bone marrow is very rare, every 10 5 ～10 6 Only 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. The latest 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 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 24/9/2008) discloses an invention entitled "placental mesenchymal stem cell isolation and in vitro amplification culture method"; CN101693884a (chinese patent application No. 200910117522.9, published 2010, 4 months and 14 days) entitled "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 titled "method for extracting original mesenchymal stem cells from placenta and serum-free amplification". In addition, chinese patent application No. 201210044648X discloses a method for isolating mesenchymal stem cells from placenta. These processes are to be further improved in terms of purity and/or recovery of the extract.
Mesenchymal stem cells, one of the adult stem cells, are derived from the early-developing mesoderm and are of great interest because of their high self-renewal, immunoregulatory and multipotential differentiation potential. Mesenchymal stem cells are widely present in various tissues throughout the body, in particular bone marrow, adipose tissue, cord blood. Mesenchymal stem cells in clinical studies are mainly derived from bone marrow. The traditional method is to obtain stem cells from bone marrow under general anesthesia or intraspinal anesthesia, but only 100-1000 colony generating units of mesenchymal stem cells can be obtained from every milliliter of bone marrow, and the clinically needed cell number can be obtained only through in vitro amplification.
In 2003, mitchell et al first confirmed that mesenchymal stem cells extracted from umbilical cord have multipotentiality, and then several scholars isolated fibroblast-like cells from Wharton's jelly of umbilical cord and confirmed that they have self-renewal, proliferation and multipotentiality, and named human umbilical cord mesenchymal stem cells (hUC-MSCs). The human umbilical cord mesenchymal stem cells are mesenchymal stem cells existing in the umbilical cord, the umbilical cord is taken as a delivery waste, compared with other mesenchymal stem cells, the human umbilical cord mesenchymal stem cells have sub-totipotent differentiation potential, are simple to obtain, rich in source, about twice as fast as other stem cells in a cultivation process, uniform in growth, and have many excellent characteristics as seed cells, such as high proliferation activity, low immunogenicity, no tumorigenicity and the like, sufficient in source, no ethical problems and the like. The potential of human umbilical cord mesenchymal stem cells in regenerative medicine and tissue engineering has been of great interest, and has advantages over bone marrow derived MSCs, in that the human umbilical cord surface is an amniotic membrane-coated epithelium comprising two arteries and a vein, and a mucous-like connective tissue (called wharton's jelly, WJ) is surrounded by umbilical cord blood vessels.
The source of umbilical cord mesenchymal stem cells comprises amniotic membrane, amnion lower layer, wharton's jelly, umbilical cord blood vessel peripheral tissue and umbilical cord blood. At present, the mesenchymal stem cells from the source of Wharton's jelly are widely applied clinically. At present, the research aspect of human umbilical cord mesenchymal stem cells has defects. The method mainly comprises the following steps: (1) After the umbilical cord is collected, the umbilical cord is quiet, the arterial blood is coagulated, the difficulty of umbilical cord treatment is increased, and the chance of stem cell pollution is increased due to excessive blood cells; (2) The tissue adherence method is to cut the umbilical cord into small tissue blocks which are directly adhered to a culture medium, and primary cells can be obtained generally for about 15 days; the disadvantage of this method is that the cycle is long; the tissue block is easy to float, so that the tissue block loses the capability of growing cells, the number of the cells is reduced, and in addition, the purity of the cells is insufficient; (3) Collagenase and trypsin are mostly used together in the enzyme digestion method, although the period is short, the cost is high, the conditions are not easy to master, cells can be damaged if the normal-temperature digestion time is long, liquid is viscous if the digestion time is short, enough cells are difficult to obtain through centrifugation, and the safety risk of anaphylactic reaction and cross infection caused by animal-derived protein in the clinical application process is increased.
In recent years, umbilical cord perivascular stem cells are considered to be progenitor cells of mesenchymal stem cells due to high proliferation activity, high clonogenic power and multidirectional differentiation potential, and have wide clinical application prospects. In 2013, tsang WP et al indicated that CD146+ pericytes could be the source of bone regeneration cells.
CN105695401A (CN 201610189133.7) discloses a preparation and preservation method of umbilical cord vein artery and vein perivascular stem cells, which is characterized in that umbilical cord perivascular derived mesenchymal stem cells with higher purity are obtained by culturing through a cell culture solution (DMEM low sugar, 10% fetal bovine serum and 1% streptomycin double antibody) compared with a common patch method; does not adopt digestive enzyme, avoids anaphylactic reaction and cross infection caused by animal-derived protein, and has higher positive rate than the mesenchymal stem cell CD146 derived from Wharton's jelly.
It is expected that there is a new source of human mesenchymal stem cells, for example, mesenchymal stem cells isolated from blood vessels of placenta, but no method for isolating mesenchymal stem cells from blood vessels of placenta has been found in the prior art. Therefore, the art still expects to be able to successfully isolate mesenchymal stem cells from the blood vessels of the placenta.
Disclosure of Invention
The invention aims to solve the defects of the existing method for obtaining the placenta mesenchymal stem cell resource, and provides a practical, simple and efficient method for separating the mesenchymal stem cells from the blood vessel of the placenta and optionally establishing a stem cell bank. Meanwhile, another object of the present invention is to provide a digestive enzyme composition for the above method for isolating mesenchymal stem cells from placental blood vessels. The inventors have found that by using a specific method of operation and a specific formulation of the digestive enzyme composition, high cell purity and/or high cell recovery can be obtained. The present invention has been completed based on this finding.
To this end, the present invention provides, in a first aspect, a digestive enzyme composition for use in a method for isolating placental mesenchymal stem cells from placental blood vessels, the digestive enzyme composition being a tissue-digesting enzyme-containing PBS buffer, the tissue-digesting enzyme-containing PBS buffer being a PBS buffer to which one or more digestive enzymes selected from the group consisting of: dispase, pancreatin, deoxyribonuclease I (DNase I), collagenase II, collagenase IV, hyaluronidase. In one embodiment, the digestive enzyme composition comprises the following digestive enzymes: deoxyribonuclease I (DNase I), collagenase II, collagenase IV.
The digestive enzyme composition according to the first aspect of the present invention is a PBS buffer containing the digestive enzyme. In one embodiment, the PBS buffer containing the digestive enzymes comprises: 0.1-0.3% such as 0.2% collagenase II, 0.1-0.2% such as 0.15% collagenase IV, 0.05-0.15% such as 0.1% DNase I.
The digestive enzyme composition according to the first aspect of the present invention is a PBS buffer containing the digestive enzyme. In one embodiment, the PBS buffer containing the digestive enzymes comprises: 0.2% collagenase II, 0.15% collagenase IV, 0.1% DNase I.
The digestive enzyme composition according to the first aspect of the present invention, wherein said method of isolating placental mesenchymal stem cells from placental blood vessels comprises the steps of:
(1) Soaking placenta in 75% ethanol for 30 s, and washing with PBS twice;
(2) Stripping the placenta blood vessel from the umbilical cord root of placenta, and squeezing the blood vessel with surgical forceps to remove blood stain;
(3) Cutting the blood vessel into 1-2mm ^3 fragments, cleaning the fragments with PBS, and filtering the fragments with a 300-mesh filter screen to remove residual blood stains to obtain a placenta vascular tissue;
(4) Adding mixed enzyme liquid for digestion;
(5) Adding serum to stop digestion, filtering with a 300-mesh filter screen, collecting tissue fluid, washing with PBS, and mixing the cleaning fluids;
(6) Centrifuging to obtain cell sediment, washing with PBS, centrifuging to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basal medium, sampling, and counting the number and the survival rate of nucleated cells; the obtained cells are frozen by using a freezing protective solution so as to be revived and cultured before use, or the obtained cells are subjected to the following steps;
(7) Inoculating the cells into a T75 culture flask, and adding a complete culture medium for culture;
(8) Changing the culture solution every 3 days in the culture process until the cell fusion rate reaches more than 80%, and carrying out passage to obtain P1 generation of placenta mesenchymal stem cells;
and optionally one or more of the following steps:
(9) Performing cell identification and/or detection (e.g., including, but not limited to, adipogenic, osteogenic and chondrogenic, flow assay, HLA identification, cellular activity, cellular contamination, genetic disease, HLA-ABC/DR match) on the placental mesenchymal stem cells obtained in step (8);
(10) Freezing and storing the placenta mesenchymal stem cells obtained in the step (8) after passage in liquid nitrogen;
(11) Establishing a database of placental stem cells comprising the above information, and correlating the database with the cryopreserved cells of step (10).
The digestive enzyme composition according to the first aspect of the present invention, wherein the mixed enzyme solution in the step (4) is a PBS buffer solution to which the mixed enzyme is added.
The digestive enzyme composition according to the first aspect of the present invention, wherein the mixed enzyme solution in the step (4) comprises: 0.1-0.3% such as 0.2% collagenase II, 0.1-0.2% such as 0.15% collagenase IV, 0.05-0.15% such as 0.1% DNase I.
The digestive enzyme composition according to the first aspect of the present invention, wherein the mixed enzyme solution is added in step (4) for digestion for 0.5 to 2 hours, for example, for digestion for 1 hour.
The digestive enzyme composition according to the first aspect of the invention, the centrifugation in step (6) is for example centrifugation at 1500rpm for 5min.
The digestive enzyme composition according to the first aspect of the invention, wherein the seeding density in step (7) is 0.5 to 2x10^5/cm ^2, for example, the seeding density is 1x10^5/cm ^2.
According to the digestive enzyme composition of the first aspect of the present invention, in the step (7), the complete medium is composed of: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF).
The digestive enzyme composition according to the first aspect of the invention, in step (7), the culturing is at 37 ℃,5% 2 Culturing in an incubator.
According to the digestive enzyme composition of the first aspect of the present invention, in the step (9), the cell activity is detected by counting the number of viable cells before and after the cryopreservation using trypan blue staining method.
According to the digestive enzyme composition of the first aspect of the present invention, in the step (9), the cell contamination detection is to detect whether the cells are contaminated with fungi and bacteria by 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 two-half, hepatitis C, AIDS virus, cytomegalovirus, EB virus and syphilis, hbsAg, hbsAb, HBcAb, hbeAg, hbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.
According to the digestive enzyme composition of the first aspect of the present invention, in the step (9), the genetic disease detection is to detect the presence of a genetic disease in the cryopreserved cells by using a molecular genetic method.
According to the digestive enzyme composition of the first aspect of the present invention, in step (9), said HLA-ABC/DR match is a phenotype of HLA-ABC/DR of the test cell.
According to the digestive enzyme composition of the first aspect of the present invention, in step (10), the placental mesenchymal stem cells are frozen in liquid nitrogen through a temperature-programmed process.
According to the digestive enzyme composition of the first aspect of the present invention, in step (10), the placental mesenchymal stem cells are present in a cell cryopreservation solution. In one embodiment, the cell culture medium comprises 50% low carbohydrate DMEM medium, 40% FBS, 10% dimethyl sulfoxide.
The digestive enzyme composition according to the first aspect of the invention, in step (11), comprises in the database all relevant data to the preserved cells, including but not limited to: the result of detecting the biological characteristics of the cells, the result of identifying the multipotentiality of the cells, the result of the molecular genetic diagnosis of the cells, and the details of the fetus and the parents.
Further, the second aspect of the present invention provides a method for isolating mesenchymal stem cells from blood vessels of a placenta, the method comprising the steps of:
(1) Soaking placenta hominis in 75% ethanol, sterilizing for 30 s, and washing with PBS twice;
(2) Stripping the placenta blood vessel from the umbilical cord root of placenta, and squeezing the blood vessel with surgical forceps to remove blood stain;
(3) Cutting the blood vessel into 1-2mm ^3 fragments, cleaning the fragments with PBS, and filtering the fragments with a 300-mesh filter screen to remove residual blood stains to obtain a placenta vascular tissue;
(4) Adding mixed enzyme solution for digestion;
(5) Adding serum to stop digestion, filtering with a 300-mesh filter screen, collecting tissue fluid, washing with PBS, and mixing the cleaning fluids;
(6) Centrifuging to obtain cell sediment, washing with PBS, centrifuging to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basal medium, sampling, and counting the number and the survival rate of nucleated cells; the obtained cells are frozen by using a freezing protective solution so as to be revived and cultured before use, or the obtained cells are subjected to the following steps;
(7) Inoculating the cells into a T75 culture flask, and adding a complete culture medium for culture;
(8) Changing the culture solution every 3 days in the culture process until the cell fusion rate reaches more than 80%, and carrying out passage to obtain P1 generation of placenta mesenchymal stem cells;
and optionally one or more of the following steps:
(9) Performing cell identification and/or detection (e.g., including, but not limited to, adipogenic, osteogenic and chondrogenic, flow assay, HLA identification, cellular activity, cellular contamination, genetic disease, HLA-ABC/DR match) on the placental mesenchymal stem cells obtained in step (8);
(10) Freezing and storing the placenta mesenchymal stem cells obtained in the step (8) after passage in liquid nitrogen;
(11) Establishing a database of placental stem cells comprising the above information, and correlating the database with the cryopreserved cells of step (10).
The method according to the second aspect of the present invention, wherein the mixed enzyme solution in step (4) is a PBS buffer solution to which the mixed enzyme is added. That is, the mixed enzyme solution is prepared by using PBS buffer solution as a solution preparation medium, and the corresponding type and amount of digestive enzyme is supplemented and added, and other substances can be added on the basis.
The method according to the second aspect of the present invention, wherein the mixed enzyme solution in the step (4) comprises: 0.1-0.3%, e.g. 0.2% collagenase II, 0.1-0.2%, e.g. 0.15% collagenase IV, 0.05-0.15%, e.g. 0.1% DNase I.
The method according to the second aspect of the present invention, wherein the mixed enzyme solution is added in the step (4) for digestion for 0.5 to 2 hours, for example, for digestion for 1 hour.
According to the method of the second aspect of the invention, in step (6) the centrifugation is for example at 1000 to 2000rpm, for example 1500rpm, for 3 to 7min, for example 5min.
According to the method of the second aspect of the invention, the seeding density in step (7) is 0.5-2x10 ^5/cm ^2, for example the seeding density is 1x10^5/cm ^2.
According to the method of the second aspect of the present invention, in step (7), the complete medium has a composition of: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF).
According to the method of the second aspect of the present invention, in step (7), the culturing is effected at 37 ℃ 5% 2 Culturing in an incubator.
According to the method of the second aspect of the present invention, in the step (9), the cell activity is detected by counting the number of viable cells before and after cryopreservation using a trypan blue staining method.
According to the method of the second aspect of the present invention, in step (9), the cell contamination detection is to detect whether or not 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 two-in-two, hepatitis C, AIDS virus, cytomegalovirus, EB virus and syphilis, hbsAg, hbsAb, HBcAb, hbeAg, hbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST.
According to the method of the second aspect of the present invention, in step (9), the genetic disease detection is a method of detecting the presence or absence of a genetic disease in the cryopreserved cells by using molecular genetics.
According to the method of the second aspect of the invention, in step (9), the HLA-ABC/DR matching is carried out by assaying the cell for HLA-ABC/DR phenotype.
According to the method of the second aspect of the present invention, in step (10), the placental mesenchymal stem cells are frozen in liquid nitrogen through a temperature-programmed process.
According to the method of the second aspect of the present invention, in step (10), the placental mesenchymal stem cells are present in a cell cryopreservation solution. In one embodiment, the cell culture medium comprises 50% low carbohydrate DMEM medium, 40% FBS, 10% dimethyl sulfoxide.
According to the method of the second aspect of the invention, in step (11), 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, a placental mesenchymal stem cell isolated from a placental blood vessel is obtained. Thus in a third aspect the present invention provides a placental mesenchymal stem cell isolated from placental blood vessels.
Placental mesenchymal stem cells according to a third aspect of the invention, obtained according to the method of any one of the embodiments of the first aspect of the invention.
The placental mesenchymal stem cells according to the third aspect of the invention have a cell purity of greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 90% after more than 1 passage.
The present invention is further described below. The documents cited in the present application, and the documents cited in the documents, are incorporated herein by reference in their entirety.
In the present invention, any of the technical features in any of the technical aspects of the present invention are equally applicable to any of the embodiments of any of the technical aspects of the present invention, as long as they do not cause contradiction, and such mutual application may be appropriately modified as necessary.
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 explicitly indicated otherwise. In addition, the present invention relates to a method of isolating placental mesenchymal stem cells from placental blood vessels, and more specifically, the placental mesenchymal stem cells of the present invention refer to placental mesenchymal stem cells derived from placental blood vessels.
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.
According to any aspect of the invention, 0.02-0.05% of sodium glutamate and 0.05-0.1% of sodium alginate are further added into the mixed enzyme solution, for example, the mixed enzyme solution comprises: 0.1 to 0.3% such as 0.2% collagenase II, 0.1 to 0.2% such as 0.15% collagenase IV, 0.05 to 0.15% such as 0.1% DNase I, 0.02 to 0.05% such as 0.03% sodium glutamate and 0.05 to 0.1% such as 0.075% sodium alginate; for example, the mixed enzyme solution may contain about 0.02%, about 0.03%, about 0.04%, or about 0.05% sodium glutamate and about 0.05%, about 0.06%, about 0.075%, or about 0.1% sodium alginate. According to any aspect of the present invention, 0.02-0.05% of sodium glutamate and 0.01-0.05% of ammonium alginate are further added to the mixed enzyme solution, for example, the mixed enzyme solution comprises: 0.1-0.3%, e.g. 0.2% collagenase II, 0.1-0.2%, e.g. 0.15% collagenase IV, 0.05-0.15%, e.g. 0.1% deoxyribonuclease I, 0.02-0.05%, e.g. 0.03% sodium glutamate and 0.01-0.05%, e.g. 0.025% ammonium alginate; for example, the mixed enzyme solution may contain about 0.02%, about 0.03%, about 0.04%, or about 0.05% sodium glutamate and about 0.01%, about 0.02%, about 0.025%, or about 0.05% ammonium alginate. It has been surprisingly found that the simultaneous addition of small amounts of sodium glutamate and sodium alginate to a tissue digesting enzyme containing PBS buffer can significantly improve cell yield, which would be of great significance. It has also been surprisingly found that the addition of small amounts of sodium glutamate and ammonium alginate simultaneously to tissue digesting enzyme-containing PBS buffer also significantly increases cell yield, and that the use of a smaller amount of ammonium alginate than sodium alginate can achieve substantially the same effect as sodium alginate, which is of great importance. This test for increasing the cell yield was carried out as follows. As shown in examples 1 to 3 below, wherein the amount of the placental vascular tissue in step (3) gives the number of nucleated cells shown in step (6), 1.93 to 2.10X 10^7 nucleated cells can be harvested per 1 g of the placental vascular tissue, and this data, as the yield of P0 generation cells, can reflect the MSC preparation efficiency of the procedure before step (6); the present inventors have attempted to replace the digestive enzymes of the present invention with several prior art-disclosed digestive enzyme formulations in method step (4) of example 1 of the present invention, resulting in P0 generation cell yields that are all less than 0.1 x10 ^7 nucleated cells/g placental vascular tissue, much lower than the present invention yields, e.g., the P0 generation cell yields of the digestive enzymes used in example 1 of chinese patent application No. 201710454588.1 (PBS buffer containing 0.1mg/mL dispase, 0.25mg/mL pancreatin, 0.25mg/mL DNase I, 1mg/mL collagenase IV, 1mg/mL hyaluronidase) are all less than 0.087 x10 ^7 nucleated cells/g placental vascular tissue; although the above-described mixed digestive enzymes of the present invention have been obtained in quite satisfactory yields, it is expected by those skilled in the art that such yields will be improved. In a supplementary example of the present invention, supplementary example A1, referring to the methods of examples 1 to 3, respectively, except that 0.03% sodium glutamate and 0.075% sodium alginate were supplementarily added to the mixed enzyme solution, it was revealed that the yields of nucleated cells in steps (1) to (6) in the three supplementary examples were 9.26X 10^7 nucleated cells/g placental vascular tissue, 9.57X 10^7 nucleated cells/g placental vascular tissue, and 9.36X 10^7 nucleated cells/g placental vascular tissue, respectively, indicating that the yields of P0 generation cells could be significantly increased by supplementarily adding sodium glutamate and sodium alginate to the mixed enzyme solution. In supplementary embodiment A2, which is a supplementary embodiment of the present invention, referring to the methods of embodiments 1-3, respectively, except that 0.03% sodium glutamate and 0.025% ammonium alginate are supplementarily added to the mixed enzyme solution, the results show that the yield of nucleated cells in steps (1) to (6) in the three supplementary embodiments are 10.32 x10 ^7 nucleated cells/g placental vascular tissue, 10.68 x10 ^7 nucleated cells/g placental vascular tissue, 10.47 x10 ^7 nucleated cells/g placental vascular tissue, respectively, indicating that the yield of P0 generation cells can be significantly increased by supplementarily adding sodium glutamate and ammonium alginate to the mixed enzyme solution; the results of this supplementary example A2 also show that the use of lower concentrations of ammonium alginate can achieve higher yields of nucleated cells than the use of higher concentrations of sodium alginate. In supplementary example B1, which is a supplementary example of the present invention, referring to the method of example 1, respectively, except that the mixed enzyme solution was supplemented with the combination of 0.02% sodium glutamate/0.075% sodium alginate, or the combination of 0.03% sodium glutamate/0.1% sodium alginate, or the combination of 0.04% sodium glutamate/0.05% sodium alginate, or the combination of 0.05% sodium glutamate/0.06% sodium alginate, it was revealed that the yields of nucleated cells in the steps (1) to (6) in the four supplementary examples were 9.33 × 10^7 nucleated cells/g placental vascular tissue, 10.37 × 10^7 nucleated cells/g placental vascular tissue, 10.02 × 10^7 nucleated cells/g placental vascular tissue, and 9.87 × 10^7 nucleated cells/g placental vascular tissue, respectively, indicating that the yields of P0 th generation cells could be significantly increased after the sodium glutamate and sodium alginate were added to the mixed enzyme solution. In supplementary example B2, which is a supplementary example of the present invention, referring to the method of example 1, respectively, except that the mixed enzyme solution was supplemented with the combination of 0.02% sodium glutamate/0.025% ammonium alginate, or supplemented with the combination of 0.03% sodium glutamate/0.05% ammonium alginate, or supplemented with the combination of 0.04% sodium glutamate/0.01% ammonium alginate, or supplemented with the combination of 0.05% sodium glutamate/0.02% ammonium alginate, the results showed that the yields of nucleated cells in the four supplementary examples from step (1) to step (6) were 10.51 × 10^7 nucleated cells/g placental vascular tissue, 11.43 × 10^7 nucleated cells/g placental vascular tissue, 11.74 × 10^7 nucleated cells/g placental vascular tissue, 10.96 × 10^7 nucleated cells/g placental vascular tissue, respectively, indicating that the yield of P0 generation cells was significantly increased after the addition of sodium glutamate and ammonium alginate to the mixed enzyme solution; the results of this supplemental example B2 also show that lower concentrations of ammonium alginate can achieve a higher yield of nucleated cells than higher concentrations of sodium alginate. In supplementary example C, which is a supplementary example of the present invention, in reference to the method of example 1, respectively, except that only 0.03% sodium glutamate, only 0.075% sodium alginate, only 0.025% ammonium alginate, or 0.03% sodium glutamate and 0.075% mannitol were additionally added to the mixed enzyme solution, it was revealed that the nucleated cell yields in the steps (1) to (6) of the four supplementary examples were 1.86 × 10^7 nucleated cells/g placental vascular tissue, 1.94 × 10^7 nucleated cells/g placental vascular tissue, 1.90 × 10^7 nucleated cells/g placental vascular tissue, 1.73 × 10^7 nucleated cells/g placental vascular tissue, respectively, indicating that the yield of P0 th generation cells could not be effectively increased by adding only the above-mentioned one supplement or by changing mannitol. The above-mentioned 1.73 x10 ^7 nucleated cells/g placental vascular tissue means the 7 th power of nucleated cells/g placental vascular tissue multiplied by 1.73 by 10, and similar expressions, particularly similar expressions related to the symbol ^ have similar meanings in the present invention.
The invention discloses a method for separating mesenchymal stem cells from placental blood vessels with high yield, and the method is used for preserving the placental mesenchymal stem cells and establishing a placental stem cell bank. Based on the summary of the conventional mesenchymal stem cells isolated and cultured, the inventors of the present invention successfully isolated and obtained a large amount of mesenchymal stem cells from the blood vessels of the placenta by using a mixed digestion tissue mass of a plurality of tissue digestive enzymes and combining with an adherent 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 prospect in clinic, placenta mesenchymal stem cells derived from placenta blood vessels 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 stem cells in the future.
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.
The invention aims to provide a practical and simple method for separating and obtaining mesenchymal stem cells from placental blood vessels with high yield and establishing the mesenchymal stem cellsA method of placental stem cell bank comprising the steps of: (1) Within four hours after delivery, the placenta was sterilized by soaking in 75% alcohol for 30 seconds under aseptic conditions, and then washed twice with PBS; (2) Stripping the placenta blood vessel from the umbilical cord root of placenta, and squeezing the blood vessel with surgical forceps to remove blood stain; (3) Cutting the blood vessel into pieces of about 1.5mm ^3, cleaning the blood vessel with PBS, and filtering the blood vessel with a 300-mesh filter screen to remove residual blood stain to obtain placenta vascular tissue; (4) Adding mixed enzyme solution (containing 0.2% collagenase II, 0.15% collagenase IV, and 0.1% DNase I, in PBS buffer) for digestion for 1h; (5) Adding serum to stop digestion, filtering with a 300-mesh filter screen, collecting tissue fluid, washing with PBS, and mixing the cleaning fluids; (6) Centrifuging (centrifuging at 1500rpm for 5 min) to obtain cell precipitate, washing with PBS, centrifuging (centrifuging at 1500rpm for 5 min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basic culture medium, sampling and counting the number and activity of nucleated cells; the obtained cells are frozen by using a freezing protective solution so as to be revived and cultured before use, or the obtained cells are subjected to the following steps; (7) Cells were inoculated into T75 flasks and cultured (37 ℃,5% CO) with complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF)) added 2 An incubator); (8) And replacing the culture solution every 3 days in the culture process until the cell fusion rate reaches more than 80%, and carrying out passage to obtain the P1 generation of the placenta mesenchymal stem cells.
The invention aims to provide a practical and simple method for separating and obtaining mesenchymal stem cells from placental blood vessels with high yield and establishing a placental stem cell bank, which comprises the following steps: (1) Within four hours after delivery, the placenta was sterilized by soaking in 75% alcohol for 30 seconds under aseptic conditions, and then washed twice with PBS; (2) Stripping the blood vessels of the placenta from the umbilical cord root of the placenta, and squeezing the blood vessels by using surgical forceps to remove blood stains; (3) Cutting the blood vessel into 1mm ^3 fragments, cleaning the fragments with PBS, and filtering the fragments with a 300-mesh filter screen to remove residual blood stains to obtain a placenta vascular tissue; (4) Adding mixed enzyme solution (containing 0.3% collagenase II, 0.1% collagenase IV, 0.15% deoxyribonuclease I, in PBS buffer) for digestion for 0.5h; (5) Adding serum to stop digestion, filtering with 300 mesh filter screen, collecting tissue fluid,washing with PBS, and combining the washing liquids; (6) Centrifuging (centrifuging at 1000rpm for 7 min) to obtain cell precipitate, washing with PBS, centrifuging (centrifuging at 1000rpm for 7 min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basic culture medium, sampling and counting the number and activity of nucleated cells; the obtained cells are frozen by using a freezing protective solution so as to be subjected to recovery culture before use, or the obtained cells are subjected to the following steps; (7) Inoculating the cells into a T75 flask (inoculation density: 2x10^5/cm ^ 2), culturing (37 ℃,5% CO) with addition of complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF)) 2 An incubator); (8) And replacing the culture solution every 3 days in the culture process until the cell fusion rate reaches more than 80%, and carrying out passage to obtain the P1 generation of the placenta mesenchymal stem cells.
The invention aims to provide a practical and simple method for separating and obtaining mesenchymal stem cells from placental blood vessels with high yield and establishing a placental stem cell bank, which comprises the following steps: (1) Within four hours after delivery, the placenta was sterilized by soaking in 75% alcohol for 30 seconds under aseptic conditions, and then washed twice with PBS; (2) Stripping the blood vessels of the placenta from the umbilical cord root of the placenta, and squeezing the blood vessels by using surgical forceps to remove blood stains; (3) Cutting the blood vessel into 2mm ^3 fragments, washing with PBS, and filtering with a 300-mesh filter screen to remove residual blood stain to obtain a placenta vascular tissue; (4) Adding mixed enzyme solution (containing 0.1% collagenase II, 0.2% collagenase IV, and 0.05% DNase I, in PBS buffer) for digestion for 2h; (5) Adding serum to stop digestion, filtering with a 300-mesh filter screen, collecting tissue fluid, washing with PBS, and mixing the cleaning fluids; (6) Centrifuging (centrifuging at 2000rpm for 3 min) to obtain cell precipitate, washing with PBS, centrifuging (centrifuging at 2000rpm for 3 min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basic culture medium, sampling and counting the number and activity of nucleated cells; the obtained cells are frozen by using a freezing protective solution so as to be revived and cultured before use, or the obtained cells are subjected to the following steps; (7) Cells were inoculated into T75 flasks and cultured (37 ℃,5% CO) with complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF)) added 2 An incubator);(8) And replacing the culture solution every 3 days in the culture process until the cell fusion rate reaches more than 80%, and carrying out passage to obtain the P1 generation of the placenta mesenchymal stem cells.
The method is simple to operate, convenient and practical, can obtain a large amount of placenta mesenchymal stem cells separated from the placenta blood vessels, has good differentiation performance, and has the capability 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 placental blood vessels, 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 the cord blood, the cell components are more immature, the source is wide, and the placenta is convenient and easy to obtain, the method has wide prospects in clinical application of stem cells.
Drawings
A, B, C, D, E of fig. 1 is the flow cytometry identification of MSC surface markers; as shown in B, D, E, the positive rates of CD73, CD90 and CD105 are all more than 98%, and as shown in C, the positive rates of CD11b, CD34, CD45, CD19 and HLA-DR are all less than 2%.
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 generally and/or specifically describes the materials used in the tests, as well as the test 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 isolation, subculture, cryopreservation of placental MSCs
(1) Within four hours after delivery, the placenta was sterilized by soaking in 75% alcohol under aseptic conditions for 30 seconds, and then washed twice with PBS;
(2) Stripping the placenta blood vessel from the umbilical cord root of placenta, and squeezing the blood vessel with surgical forceps to remove blood stain;
(3) Cutting the blood vessel into pieces of about 1.5mm ^3, washing with PBS, and filtering with a 300-mesh filter screen to remove residual blood stain to obtain the placental vascular tissue (5.8 g of placental vascular tissue is obtained from one placenta in this example);
(4) Adding mixed enzyme solution (containing 0.2% collagenase II, 0.15% collagenase IV, and 0.1% DNase I, in PBS buffer) for digestion for 1h;
(5) Adding serum to stop digestion, filtering with a 300-mesh filter screen, collecting tissue fluid, washing with PBS, and mixing the cleaning fluids;
(6) Centrifuging (centrifuging at 1500rpm for 5 min) to obtain cell precipitate, washing with PBS, centrifuging (centrifuging at 1500rpm for 5 min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basal medium, sampling, and counting the number and activity of nucleated cells (to obtain about 1.22 × 10^8 nucleated cells with activity of more than 90%); the obtained cells are frozen by using a freezing protective solution so as to be revived and cultured before use, or the obtained cells are subjected to the following steps;
(7) Cells were inoculated into T75 flasks (inoculation density: 1X10^5/cm ^ 2), cultured (37 ℃ C., 5% CO) with addition of complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF)) 2 An incubator);
(8) Changing the culture solution every 3 days in the culture process until the cell fusion rate reaches more than 80 percent (usually about 10 days), and carrying out passage to obtain the P1 generation of the placenta mesenchymal stem cells (thereby obtaining the mesenchymal stem cells with the number of 2.54 multiplied by 10^8 and the activity can reach more than 95 percent).
Then, the process as in step (8) is continued for the passage to the desired passage. The resulting MSCs were subjected to subsequent experimental assays.
Example 2 isolation, subculture, cryopreservation of placental MSCs
(1) Within four hours after delivery, the placenta was sterilized by soaking in 75% alcohol for 30 seconds under aseptic conditions, and then washed twice with PBS;
(2) Stripping the placenta blood vessel from the umbilical cord root of placenta, and squeezing the blood vessel with surgical forceps to remove blood stain;
(3) Cutting the blood vessels into 1mm ^3 fragments, washing with PBS, and filtering with a 300-mesh filter screen to remove residual blood stain to obtain the placenta vascular tissue (5.5 g of placenta vascular tissue is obtained from one placenta in the example);
(4) Adding mixed enzyme solution (containing 0.3% collagenase II, 0.1% collagenase IV, 0.15% deoxyribonuclease I, in PBS buffer) for digestion for 0.5h;
(5) Adding serum to stop digestion, filtering with a 300-mesh filter screen, collecting tissue fluid, washing with PBS, and mixing the cleaning fluids;
(6) Centrifuging (centrifuging at 1000rpm for 7 min) to obtain cell precipitate, washing with PBS, centrifuging (centrifuging at 1000rpm for 7 min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basal medium, sampling, and counting the number and activity of nucleated cells (to obtain about 1.06 × 10^8 nucleated cells with activity of more than 90%); the obtained cells are frozen by using a freezing protective solution so as to be revived and cultured before use, or the obtained cells are subjected to the following steps;
(7) Cells were inoculated into T75 flasks (inoculation density: 2x10^5/cm ^ 2), cultured (37 ℃ C., 5% CO) with addition of complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF)) 2 An incubator);
(8) Changing the culture solution every 3 days in the culture process until the cell fusion rate reaches more than 80 percent (usually about 10 days), and carrying out passage to obtain the P1 generation of the placenta mesenchymal stem cells (thereby obtaining the mesenchymal stem cells with the number of 2.18 multiplied by 10^8 and the activity can reach more than 95 percent).
Then, the process continues as in step (8) until the desired passage. The resulting MSCs were subjected to subsequent experimental assays.
Example 3 isolation, subculture, cryopreservation of placental MSCs
(1) Within four hours after delivery, the placenta was sterilized by soaking in 75% alcohol under aseptic conditions for 30 seconds, and then washed twice with PBS;
(2) Stripping the blood vessels of the placenta from the umbilical cord root of the placenta, and squeezing the blood vessels by using surgical forceps to remove blood stains;
(3) Cutting the blood vessels into 2mm ^3 fragments, washing with PBS, and filtering with a 300-mesh filter screen to remove residual blood stain to obtain placenta vascular tissue (4.7 g of placenta vascular tissue is obtained from one placenta in the example);
(4) Adding mixed enzyme solution (containing 0.1% collagenase II, 0.2% collagenase IV, and 0.05% DNase I, in PBS buffer) for digestion for 2h;
(5) Adding serum to stop digestion, filtering with a 300-mesh filter screen, collecting tissue fluid, washing with PBS, and mixing the cleaning fluids;
(6) Centrifuging (centrifuging at 2000rpm for 3 min) to obtain cell precipitate, washing with PBS, centrifuging (centrifuging at 2000rpm for 3 min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basal medium, sampling, and counting the number and activity of nucleated cells (to obtain the number of nucleated cells about 0.93 × 10^8 with activity over 90%); the obtained cells are frozen by using a freezing protective solution so as to be revived and cultured before use, or the obtained cells are subjected to the following steps;
(7) Cells were inoculated into T75 flasks (inoculation density: 0.5 x10^5/cm ^ 2) and cultured (37 ℃,5% CO) with addition of complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF)) 2 An incubator);
(8) Changing the culture solution every 3 days in the culture process until the cell fusion rate reaches more than 80 percent (usually about 10 days), and carrying out passage to obtain the P1 generation of the placenta mesenchymal stem cells (thereby obtaining the mesenchymal stem cells with the number of 1.83 multiplied by 10^8 and the activity can reach more than 95 percent).
Then, the process continues as in step (8) until the desired passage. The resulting MSCs were subjected to subsequent experimental assays.
Test example 1 biological characterization of placental MSCs
By the operation of example 1, it is possible to:
performing cell identification and/or detection (e.g., including but not limited to, adipogenesis, osteogenesis and chondrogenesis, flow detection, HLA identification, cell viability, cell contamination, genetic disease, HLA-ABC/DR match) on the placental mesenchymal stem cells obtained in step (8);
freezing and storing the placenta mesenchymal stem cells obtained in the step (8) after passage in liquid nitrogen; and/or
A database of placental stem cells is established containing the above information and associated with the cryopreserved cells.
1. Cell growth and morphological characteristics thereof
By isolated culture in example 1, fusiform adherent cells can be clearly seen under a microscope after placenta mononuclear cells are cultured for 72 hours, turbo-shaped cell clones can be formed in about 10 days, and about 80% fused adherent layers can be formed after digestion and passage. In the culture process, the cell is found to be relatively uniform in shape, high in proliferation speed and adhesion speed, easy to digest by pancreatin and passage to more than 15 generations, and the shape and growth characteristics of the cell are not obviously changed.
2. Flow cytometry identification of MSC surface markers
The cell surface markers were detected by flow cytometry from the 0 th, 1 th, 3 th and 6 th generation cells, respectively, by isolated culture in example 1, and the changes in the cell surface markers during culture were dynamically observed. Digesting and collecting cells, counting and taking 8X 10 cells 6 Each cell is divided into 16 tubes; washing with PBS once, and centrifuging at 1500rpm for 10min; discarding the supernatant, leaving 100-200 mul, and blowing and mixing the cells; adding 10 μ l each of PE-labeled CD14, CD29, CD31, CD34, CD44, CD54, CD73, CD80, CD86, CD166 antibodies and FITC-labeled CD45, CD105, HLA-ABC, HLA-DR, UEA-1 antibodies, and setting one tube as blank control; reacting at 4 ℃ in the dark for 30min; washing with PBS once, and centrifuging at 1500rpm for 10min; the supernatant of the directly labeled cells is discarded, 200 mul of PBS is added to blow and beat the cells evenly, 200 mul of 1% paraformaldehyde is fixed, the cells are placed at 4 ℃ to be detected, and the cells are detected by an upper flow cytometer within 3 days.
The flow cytometry detects the surface mark of the cell, dynamically observes the 0 th, 1 st, 3 th and 6 th generation cells, and has no obvious change. The flow detection results show that the positive rates of CD73, CD90 and CD105 are all more than 98%, the positive rates of CD11b, CD34, CD45, CD19 and HLA-DR are all less than 2%, and the specific results are shown in figure 1.
3. Flow cytometry detection of cell cycle of placental MSCs
When the cells were grown to about 80% and fused by the isolation culture in example 1, about 1X10 cells were collected by digestion 6 And washing once with PBS, adding 70% ethanol for fixation, and detecting at 4 ℃. During detection, firstly centrifuging to remove ethanol, washing with PBS once, adding RNase I500u, reacting at 37 ℃ for 30min, washing with PBS once, adding 1ml of propidium iodide (PI, with the final concentration of 50 mu g/ml), reacting at room temperature in a dark place for 20min, and detecting the DNA content of cells by a computer. The results show that the cells cultured in vitro have typical stem cell proliferation characteristics, i.e. only a few cells are in the active proliferation phase (<1.5%) and the majority of the cells are in the resting phase (1: (1)>95％)。
4. Drawing of placenta MSC growth curve and determination of logarithmic growth phase doubling time
Cells in the logarithmic growth phase were collected by the isolation culture in example 1, digested and counted, and prepared into a cell suspension (2X 10) in LG-DMEM medium at a concentration of 10% FBS 4 Per ml), inoculation of 0.5ml per well in 24-well plates, 37 ℃% 2 Culturing under saturated humidity. 3 wells are taken every day, viable cell number is counted after trypan blue staining, average value is calculated, and continuous observation is carried out for 7 days. The cell growth curve was plotted with the horizontal axis of culture time and the vertical axis of cell number. The doubling time of the cells in the logarithmic growth phase was calculated by the Patterson formula, i.e., td = Tlg2/Lg (Nt/No), td: doubling time (h), T: time taken for the cell to increase from No to Nt (h), N: the number of cells. Doubling time was calculated by plotting cell growth curves from the results of daily cell counts. As can be seen from the cell growth curves, the cells were in exponential growth phase on days 2-4.
5. Identification of placental MSC multipotentiality
(1) To becomeOsteoinduction
By isolated culture of example 1, 3 or more generations of MSCs were grown at 1X10 5 Six well plates were inoculated per well at 37 ℃ and 5% CO 2 After culturing in MSC medium for 24h under saturation humidity, DMEM-HG containing 10% of screened FBS was replaced with dexamethasone 0.1. Mu.M, ascorbyl phosphate 50. Mu.M, and beta-glycerophosphate 10mM, and the mixture was allowed to stand at 37 ℃ and 5% CO 2 Culturing under saturated humidity, changing liquid every 3 days for half a day, and inducing for 2-4 weeks. Alkaline phosphatase staining identifies osteoblast formation and Von Kossa staining identifies bone nodule formation.
After the cells are cultured for 1 week by adding 0.1 mu M of dexamethasone, 50 mu M of ascorbyl phosphate and 10mM of beta-glycerophosphate into DMEM-HG containing 10% of screened FBS, the cell morphology is obviously changed, the spindle-shaped fibroblast cells are changed into polygonal cells, and the cell peripheries are provided with filamentous protrusions similar to neuron cells and can extend to the peripheries. After the culture is continued for more than 2 weeks, calcified spots appear in the cell matrix, mineralized substances gradually appear, a multi-layer nodule structure begins to form, and obvious calcified nodules are visible after the culture is continued for 4 weeks. The alkaline phosphatase staining was strongly positive at 2 weeks, reaching more than 95%, whereas the uninduced controls were mostly negative and less than 5% were weakly positive, indicating that the cells had transformed into osteoblasts. von Kossa staining stained the calcium deposited in the bone nodules black, and a large number of black bone nodules were visible in the induced group with distinct spatial structure, while the control group did not respond positively at any time.
(2) Induction of adipogenesis
By isolated culture of example 1, 3 or more generations of MSCs were grown at 1X10 5 Inoculating in six-well plates at 37 ℃ and 5% CO 2 Culturing in MSC culture medium under saturation humidity for 24 hr, adding high-sugar DMEM containing 10% screened FBS, dexamethasone 1 μ M, indomethacin 60 μ M, IBMX 0.5mM, and insulin 5 μ g/ml, placing at 37 deg.C, and removing CO 5% 2 Culturing under saturated humidity, changing liquid half a day, inducing for 2 weeks, and dyeing with oil red to identify lipid drop formation.
Adding 1 mu M of dexamethasone, 200 mu M, IBMX.5 mM of indomethacin and 10 mu g/ml of insulin into DMEM-HG containing 10% of screened FBS, and culturing for 3 days, wherein the cells are subjected to morphological change, gradually shrink and shorten like spindle-shaped fibroblasts, and more than 90% of the cells become cubic or polygonal; after the cells are continuously cultured for 7 days, tiny lipid droplets in the cells can be seen to appear under the microscope, the lipid droplets gradually increase and fuse with the prolonging of the culture time, and when the cells are cultured for 2 weeks, the lipid droplets which fuse into clusters can be seen to fill the whole cells. Oil red O staining revealed that intracellular fat was specifically stained red.
(3) Chondrogenic induction
Through the isolation culture of example 1, 3 or more generations of cells were cultured in a manner of 2X10 cells per tube 5 The cells were dispensed into 15ml polypropylene centrifuge tubes, centrifuged at low speed to form micelles in the tubes, and then insulin, transferrin, and sodium selenite were added to DMEM-HG containing 2.5% FBS in an amount of 6.25. Mu.g/ml each, BSA 1.25. Mu.g/ml, sodium pyruvate 1mM/L, ascorbyl phosphate 37.5. Mu.g/ml, TGF-. Beta. 1 50ng/ml, at 37 ℃ and 5% CO 2 Culturing under saturated humidity, changing liquid every 3 days for half, and continuously culturing for 2 weeks.
After 2 weeks of induction, the cell micelles were smeared and stained with Oceann blue (pharmacin blue) to visualize collagen type II to form an extracellular matrix which was blue, and no blue staining was observed in the control group.
6. RT-PCR detection of placenta MSC multidirectional differentiation potential
Collecting induced cells, extracting total RNA of the cells by using Trizol reagent, performing RT-PCR by using the total RNA as a template, and performing reverse transcription and PCR operation according to the specification of an RT-PCR kit, wherein the names, sequences, sizes and specificities of primers are shown in the table 1 of CN102676451A (0086) to 0087). The results show that after in vitro induction cells express a series of specific mrnas: the cells after adipogenesis induction express PPAR-gamma, the cells after osteogenesis induction express Osteopontin (Osteopontin), and the cells after chondrogenesis induction express Collagen II (Collagen II), which shows that the obtained MSC cells have osteogenic, adipogenic and chondrogenesis differentiation capacity and accord with the accepted MSC standard.
Through the detection of a series of data indexes, the MSC separated by the method has the capacity of differentiating osteoblasts, adipocytes and chondrocytes, and the MSC obtained by the method is proved to have the characteristics of stem cells.
The placental mesenchymal stem cells obtained in example 2 and example 3 were also measured/treated according to the method of this test example, and the results were substantially the same as those of the cells obtained in example 1.
Test example 2 establishment of placental stem cell library
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 a pathogenic 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. The results of HLA detection of the cells obtained in example 1 are shown in the following table, showing that MSC is consistent with cord blood cells.
| HLA-A* | HLA-B* | HLA-DRB1* |
| 26:01，33:03 | 15:01，58:01 | 03:01，04:03 |
5. Investigation of cell origin
The details of the fetus and its parents are recorded and recorded on the record.
6. Establishment of placenta 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.
The placental mesenchymal stem cells obtained in example 2 and example 3 were also measured/treated according to the method of this test example, and the results were substantially the same as those of the cells obtained in example 1.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (12)
1. A digestive enzyme composition for use in a method of isolating placental mesenchymal stem cells from placental blood vessels, the digestive enzyme composition being a PBS buffer containing: 0.1 to 0.3 percent of collagenase II, 0.1 to 0.2 percent of collagenase IV, 0.05 to 0.15 percent of deoxyribonuclease I, 0.02 to 0.05 percent of sodium glutamate and 0.01 to 0.05 percent of ammonium alginate.
2. The digestive enzyme composition according to claim 1, wherein the digestive enzyme composition is a PBS buffer comprising: 0.2% collagenase II, 0.15% collagenase IV, 0.1% dnase I, 0.03% sodium glutamate and 0.025% ammonium alginate.
3. A method of isolating mesenchymal stem cells from blood vessels of the placenta, the method comprising the steps of:
(1) Soaking placenta in 75% ethanol for 30 s, and washing with PBS twice;
(2) Stripping the placenta blood vessel from the umbilical cord root of placenta, and squeezing the blood vessel with surgical forceps to remove blood stain;
(3) Cutting the blood vessel into pieces of 1 to 2mm 3 Washing the fragments with PBS, and filtering with a 300-mesh filter screen to remove residual blood stain to obtain placenta vascular tissue;
(4) Digesting with the digestive enzyme composition of claim 1 or 2;
(5) Adding serum to stop digestion, filtering with a 300-mesh filter screen, collecting tissue fluid, washing with PBS, and mixing the cleaning fluids;
(6) Centrifuging to obtain cell sediment, washing the cell sediment once by PBS, centrifuging to obtain original P0-generation placental mesenchymal stem cells, re-suspending by a DMEM-F12 basic culture medium, sampling and counting the number and the survival rate of nucleated cells; the obtained cells are frozen by using a freezing protective solution so as to be subjected to recovery culture before use, or the obtained cells are subjected to the following steps;
(7) Inoculating the cells into a T75 culture flask, and adding a complete culture medium for culture;
(8) And replacing the culture solution every 3 days in the culture process until the cell fusion rate reaches more than 80%, and carrying out passage to obtain the P1 generation of the placenta mesenchymal stem cells.
4. The method according to claim 3, further comprising the steps of:
(9) Performing cell identification and detection on the placenta mesenchymal stem cells obtained in the step (8);
(10) Freezing and storing the placenta mesenchymal stem cells obtained in the step (8) after passage in liquid nitrogen;
(11) Establishing a database of placental stem cells comprising the above information, and correlating the database with the cryopreserved cells of step (10).
5. The method according to claim 3, wherein the digestive enzyme composition is added in step (4) for digestion for 0.5 to 2h.
6. A method according to claim 3, wherein in step (6) the centrifugation is carried out at 1000 to 2000rpm for 3 to 7min.
7. The method according to claim 3, wherein the inoculation density in step (7) is 0.5 to 2x10 5 /cm 2 。
8. The method according to claim 3, wherein in step (7), the complete medium has a composition of: DMEM-F12+15% FBS +10ng/ml basic fibroblast growth factor.
9. The method according to claim 3, wherein in the step (7), the culturing is carried out at 37 ℃ and 5% CO 2 Culturing in an incubator.
10. The method according to claim 4, wherein in the step (9), the cell activity assay is carried out by counting the number of viable cells before and after the cryopreservation using the trypan blue staining method.
11. The method according to claim 4, wherein in the step (9), a cell contamination test is performed by detecting whether the cells are contaminated with fungi and bacteria using a small amount of cell culture.
12. The method according to claim 4, wherein in step (9), a genetic disease detection is carried out by detecting the presence or absence of a genetic disease in the cryopreserved cells by using a molecular genetic method.
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