CN109234229B - Method for separating mesenchymal stem cells from placental blood vessels and digestive enzyme composition used in same - Google Patents
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Abstract
The present invention relates to a method for isolating mesenchymal stem cells from placental blood vessels and a digestive enzyme composition used therefor. The method comprises the following steps: sterilizing placenta with ethanol; stripping placental blood vessels from the placenta; cutting into pieces, washing with PBS, and filtering to remove residual blood stain to obtain placenta vascular tissue; adding mixed enzyme solution for digestion; stopping digestion, filtering and collecting tissue fluid; centrifuging the obtained cells to precipitate original placenta mesenchymal stem cells (P0 generation), re-suspending with DMEM-F12 basic culture medium, sampling and counting the number and the survival rate of nucleated cells; and (3) freezing the obtained cells by using a freezing protection solution so as to recover and culture the cells before use, or continuing to passage and/or carrying out cell identification and/or detection, freezing, bank building and other operations on the obtained mesenchymal stem cells. The method can effectively improve the efficiency of separating the mesenchymal stem cells from the blood vessels of the placenta.
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 having a unique formulation according to 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 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. mesenchyme stem cells. Jthop Res.1991,9:641-650.Pittenger MF, Mackay AM, Beck, et al. multilineagent sensory 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.
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 required 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 human umbilical cord mesenchymal stem cells have superiority compared to bone marrow-derived MSCs, and the surface of human umbilical cord is an amniotic membrane-coated epithelium comprising two arteries and a vein, and the blood vessels of umbilical cord are surrounded by a mucus-like connective tissue (called wharton's jelly, WJ).
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 in an 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, the 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 proteins 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 (CN201610189133.7) discloses a preparation and preservation method of umbilical vein artery and vein perivascular stem cells, which is characterized in that umbilical vein perivascular mesenchymal stem cells with higher purity are obtained by culturing cell culture solution (DMEM low sugar, 10% fetal bovine serum and 1% streptomycin double antibody) than 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.
Accordingly, in a first aspect the present invention provides a method of isolating mesenchymal stem cells from blood vessels of a 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 1-2 mm ^3 fragments, cleaning the blood vessel with PBS (phosphate buffer solution), and filtering the blood vessel 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 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 the 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 detection, H L a identification, cellular activity, cellular contamination, genetic disease, H L a-ABC/DR typing) 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 first 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 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 method according to the first aspect of the present invention, wherein the mixed enzyme solution is added in the step (4) for digestion for 0.5-2 hours, for example, digestion for 1 hour.
According to the process of the first aspect of the present invention, in step (6), the centrifugation is, for example, at 1000 to 2000rpm, for example 1500rpm, for 3 to 7min, for example 5 min.
According to the method of the first aspect of the present invention, the seeding density in step (7) is 0.5-2 x10^5/cm ^2, for example, the seeding density is 1x10^5/cm ^ 2.
According to the method of the first 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 first aspect of the present invention, in the step (7), the culturing is carried out at 37 ℃ with 5% CO2Culturing in an incubator.
According to the method 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 cryopreservation using trypan blue staining.
According to the method 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, hepatitis C, HIV, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.
According to the method of the first 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 first aspect of the invention, in step (9), said H L A-ABC/DR typing is detection of the H L A-ABC/DR phenotype of the cell.
According to the method 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 method 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 sugar DMEM medium, 40% FBS, 10% dimethyl sulfoxide.
According to the method of the first 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 first aspect of the present invention, a placental mesenchymal stem cell isolated from placental blood vessels is obtained. Thus in a second aspect the present invention provides placental mesenchymal stem cells isolated from placental blood vessels.
Placental mesenchymal stem cells according to a second aspect of the invention, obtained according to the method according to any one of the embodiments of the first aspect of the invention.
The placental mesenchymal stem cells according to the second 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.
Further, a third aspect of the present invention provides 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 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 third 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 third 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 third 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 1-2 mm ^3 fragments, cleaning the blood vessel with PBS (phosphate buffer solution), and filtering the blood vessel 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 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 the 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 detection, H L a identification, cellular activity, cellular contamination, genetic disease, H L a-ABC/DR typing) 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 third 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 third 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.
According to the digestive enzyme composition of the third aspect of the present invention, in the step (4), the mixed enzyme solution is added for digestion for 0.5 to 2 hours, for example, digestion for 1 hour.
According to the digestive enzyme composition of the third aspect of the present invention, the centrifugation in step (6) is, for example, centrifugation at 1500rpm for 5 min.
The digestive enzyme composition according to the third aspect of the present 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 third 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).
According to the digestive enzyme composition of the third aspect of the present invention, in the step (7), the cultivation is carried out at 37 ℃ with 5% CO2Culturing in an incubator.
According to the digestive enzyme composition of the third 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 a trypan blue staining method.
According to the digestive enzyme composition of the third 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, hepatitis C, HIV, 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 third aspect of the present invention, in the step (9), the genetic disease detection is to detect whether the genetic disease exists in the cryopreserved cells by using a molecular genetic method.
According to the digestive enzyme composition of the third aspect of the present invention, in step (9), said H L A-ABC/DR match is a test cell H L A-ABC/DR phenotype.
According to the digestive enzyme composition of the third 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 third aspect of the present invention, in the step (10), the placental mesenchymal stem cells are present in a cell cryopreservation solution. In one embodiment, the cell culture medium comprises 50% low sugar DMEM medium, 40% FBS, 10% dimethyl sulfoxide.
According to the digestive enzyme composition of the third aspect of the present invention, in step (11), the database includes data related to all 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.
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 otherwise specifically indicated. 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.
The results of the procedures for supplementing sodium glutamate with 0.02-0.05% sodium alginate and 0.05-0.1% sodium alginate in the mixed enzyme solution according to any one aspect of the present invention, for example, the procedures for supplementing sodium alginate with 0.1-0.3%, for example, 0.2% collagenase II, 0.1-0.2%, for example, 0.15% collagenase IV, 0.05-0.15%, for example, 0.1% deoxyribonuclease I, 0.02-0.05%, for example, 0.03% sodium glutamate, and 0.05-0.1%, for example, 0.075% sodium alginate in the mixed enzyme solution according to any one of the procedures for supplementing sodium alginate with 0.7-0.7/7, or the procedures for supplementing sodium alginate with the same or similar procedures for supplementing sodium alginate with no more than 0.7, 7, or no more than 7, 8, 7, 8, 7, 8, 7, 8, 7, 8, 7, 8.
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 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 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 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 1 h; (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 (at 1500rpm for 5min) to obtainPrecipitating cells, washing the cells by PBS, centrifuging the cells (centrifuging the cells for 5min at 1500 rpm) to obtain original placenta mesenchymal stem cells (P0 generation), re-suspending the cells 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 revived and cultured before use, or the obtained cells are subjected to the following steps; (7) the cells were inoculated into a T75 flask, and cultured in a complete medium (DMEM-F12 +15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF)) at 37 ℃ with 5% CO2An incubator); (8) and (3) changing the culture solution once 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 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 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 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 0.5 h; (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 7min) to obtain cell precipitate, washing with PBS, centrifuging (centrifuging at 1000rpm for 7min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basic culture medium, sampling and counting the number and activity 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) the cells were inoculated into T75 flasks (inoculation density: 2x10^5/cm ^2), and cultured (37 ℃, 5% CO) with the addition of complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF))2An incubator); (8) changing the culture solution every 3 days in the culture process until the cell fusion rateReaching more than 80 percent, and carrying out passage to obtain the P1 generation of 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 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 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 2 h; (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 3min) to obtain cell precipitate, washing with PBS, centrifuging (centrifuging at 2000rpm for 3min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basic culture medium, sampling and counting the number and activity 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) the cells were inoculated into a T75 flask, and cultured in a complete medium (DMEM-F12 +15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF)) at 37 ℃ with 5% CO2An incubator); (8) and (3) changing the culture solution once 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 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 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
A, B, C, D, E in FIG. 1 is the result of identifying MSC surface marker by flow cytometry, wherein, as shown in FIG. B, D, E, the positive rates of CD73, CD90 and CD105 are all more than 98%, and as shown in FIG. C, the positive rates of CD11b, CD34, CD45, CD19 and H L A-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 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 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 vessel into pieces of about 1.5mm ^3, washing with PBS, and filtering with 300 mesh filter to remove residual blood stain to obtain placenta vascular tissue (5.8 g of placenta 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 1 h;
(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 5min) to obtain cell sediment, washing with PBS, centrifuging (centrifuging at 1500rpm for 5min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basic medium, sampling and counting the number and the activity rate of nucleated cells (obtaining the number of nucleated cells about 1.22 × 10^8, the activity is more than 90 percent), freezing the obtained cells with a freezing protection solution so as to resuscitate and culture before use, or performing the following steps on the obtained cells;
(7) the cells were inoculated into T75 flasks (inoculation density: 1X10^5/cm ^2), and cultured (37 ℃, 5% CO) with the addition of complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF))2An 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 placenta mesenchymal stem cells (thereby obtaining the mesenchymal stem cells with the number of 2.54 × 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 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 0.5 h;
(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 7min) to obtain cell sediment, washing with PBS, centrifuging (centrifuging at 1000rpm for 7min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basic medium, sampling and counting the number and the activity rate of nucleated cells (obtaining the number of nucleated cells about 1.06 × 10^8, the activity is more than 90 percent);
(7) the cells were inoculated into T75 flasks (inoculation density: 2x10^5/cm ^2), and cultured (37 ℃, 5% CO) with the addition of complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF))2An 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 placenta mesenchymal stem cells (thereby obtaining the mesenchymal stem cells with the number of 2.18 × 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 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 2mm ^3 fragments, washing with PBS, and filtering with a 300-mesh filter screen to remove residual blood stain to obtain the 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 2 h;
(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 3min) to obtain cell sediment, washing with PBS, centrifuging (centrifuging at 2000rpm for 3min) to obtain original placenta mesenchymal stem cells (P0 generation), resuspending with DMEM-F12 basic medium, sampling and counting the number and the activity rate of nucleated cells (obtaining the number of nucleated cells about 0.93 × 10 < 10 > 8, wherein the activity is more than 90 percent);
(7) the cells were inoculated into T75 flasks (inoculation density: 0.5 x10^5/cm ^2), and cultured in complete medium (composition: DMEM-F12+15% FBS +10ng/ml Basic Fibroblast Growth Factor (BFGF)) at 37 ℃ with 5% CO2An 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 placenta mesenchymal stem cells (thereby obtaining the mesenchymal stem cells with the number of 1.83 × 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, adipogenic, osteogenic and chondrogenic, flow detection, H L a identification, cellular activity, cellular contamination, genetic disease, H L a-ABC/DR typing) 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. During the culture process, the cell is found to be relatively uniform in shape, high in proliferation speed and adhesion speed, easy to be digested by pancreatin, and free from obvious change in shape and growth characteristics after passage to more than 15 generations.
2. Flow cytometry identification of MSC surface markers
The separation culture of example 1, 0 th, 1 st, 3 th and 6 th generation cells were taken, the cell surface markers were detected by flow cytometry, the change of the cell surface markers during the culture was observed dynamically, the cells were collected by digestion, and 8 × 10 was taken after counting6And (2) dividing each cell into 16 tubes, washing the cells once by PBS, centrifuging the cells at 1500rpm for 10min, removing the supernatant, leaving 100-200 mul of the supernatant, blowing and mixing the cells uniformly, adding 10 mul of each of PE-labeled CD14, CD29, CD31, CD34, CD44, CD54, CD73, CD80, CD86 and CD166 antibodies and FITC-labeled CD45, CD105, H L A-ABC, H L A-DR and UEA-1 antibodies, setting a tube as a blank control, reacting the cells in a dark place for 30min at 4 ℃, washing the cells once by PBS and centrifuging the cells at 1500rpm for 10min, directly labeling the cells, removing the supernatant, adding 200 mul of PBS, blowing and mixing the cells uniformly, fixing 200 mul of 1% paraformaldehyde, placing the cells at 4 ℃ for testing, and testing the cells by an upflow cytometer within 3 days.
The flow cytometry is used for detecting surface marks of cells, the cells of 0 th, 1 st, 3 th and 6 th generations are dynamically observed, and no obvious change is caused, the flow detection result shows that the positive rates of CD73, CD90 and CD105 are all more than 98%, the positive rates of CD11b, CD34, CD45, CD19 and H L A-DR are all less than 2%, and the specific result is 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 of example 1, about 1 × 10 cells were collected by digestion6And 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 I500 u, reacting at 37 ℃ for 30min, washing with PBS once, adding 1ml of propidium iodide (PI, final concentration is 50 mu g/ml), reacting at room temperature in a dark place for 20min, and detecting the content of cell DNA by a computer. Results displayThe cells cultured in vitro have the typical proliferation characteristics of stem cells, 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 were collected in the logarithmic growth phase by the isolated culture of example 1, digested and counted, and prepared into a cell suspension in L G-DMEM medium containing 10% FBS (2 × 10)4Per ml), 0.5ml of each well of a 24-well plate was inoculated with 5% CO at 37 ℃2Taking 3 wells per day, counting the number of living cells after trypan blue staining, calculating the average value, and continuously observing for 7 days, taking the culture time as the horizontal axis and the cell number as the vertical axis, and drawing a cell growth curve.
5. Identification of placental MSC multipotentiality
(1) Osteogenic induction
Isolated culture of 3 or more generations of MSCs according to example 1, 1 × 105Inoculating six-well plate in 37 deg.C and 5% CO2Culturing in MSC culture medium under saturation humidity for 24 hr, adding 10% DMEM-HG containing screened FBS, dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, and β -glycerol phosphate 10mM, placing at 37 deg.C and 5% CO2Culturing 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 culture is continued for more than 2 weeks, calcification spots appear in cell matrixes, mineralizers gradually appear and form a multi-layer nodule structure, and after the culture is continued for 4 weeks, obvious calcification nodules are visible, alkaline phosphatase staining shows a strong positive reaction at 2 weeks and reaches more than 95 percent, while the uninduced control group is mostly negative, and only less than 5 percent shows weak positive, which indicates that the cells are transformed to osteoblasts.
(2) Induction of adipogenesis
Isolated culture of 3 or more generations of MSCs according to example 1, 1 × 105Inoculating to six-well plate at 37 deg.C and 5% CO2Culturing 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 0.5.5 mM, and insulin 5 μ g/ml, placing at 37 deg.C and 5% CO2Culturing under saturated humidity, changing liquid half a day, inducing for 2 weeks, and dyeing with oil red to identify lipid drop formation.
Adding dexamethasone 1 μ M, indomethacin 200 μ M, IBMX 0.5.5 mM, and insulin 10 μ g/ml into DMEM-HG containing 10% of screened FBS, culturing for 3 days to obtain cells with changed morphology, gradually shrinking and shortening spindle-shaped fibroblast, and making more than 90% of cells into cubic or polygonal shape; 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 accordance with 2 × 10 per tube5The cells were dispensed into 15ml polypropylene centrifuge tubes, centrifuged at low speed to form micelles in the tubes, and insulin, transferrin, and sodium selenite were added to DMEM-HG containing 2.5% FBS at a concentration of 6.25. mu.g/ml each, BSA at a concentration of 1.25. mu.g/ml, sodium pyruvate at a concentration of 1 mM/L, ascorbyl phosphate at a concentration of 37.5. mu.g/ml,TGF-β150ng/ml, placing at 37 deg.C and 5% CO2Culturing 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, performing reverse transcription and PCR operation according to the specification of an RT-PCR kit, wherein the name, sequence, size and specificity of a primer are shown in the table 1 of [0086] to [0087] of CN 102676451A. 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 capacities and meet the recognized 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 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. H L A-ABC/DR match
Cells were tested for the H L A-ABC/DR phenotype and reported in the protocol example 1 cells were tested for H L A as 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 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.
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 (14)
1. 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 1-2 mm3Washing the fragments with PBS, and filtering with a 300-mesh filter screen to remove residual blood stain to obtain placenta vascular tissue;
(4) adding mixed enzyme solution for digestion; the mixed enzyme solution is PBS buffer solution added with mixed enzyme, and the mixed enzyme consists of the following three components: 0.1-0.3% of collagenase II, 0.1-0.2% of collagenase IV, 0.05-0.15% of deoxyribonuclease I, and the mixed enzyme solution also comprises 0.02-0.05% of sodium glutamate and 0.05-0.1% of sodium alginate;
(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 placenta mesenchymal stem cells, resuspending the placenta mesenchymal stem cells by a 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 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 the P1 generation of placenta mesenchymal stem cells;
and optionally one or more of the following steps:
(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 information of the above step (9) cell identification and detection, and correlating the database with the cryopreserved cells of step (10).
2. The method according to claim 1, wherein the mixed enzyme solution in the step (4) is composed of three of: 0.2% collagenase II, 0.15% collagenase IV, 0.1% DNase I.
3. The method according to claim 1, wherein the mixed enzyme solution is added in the step (4) for digestion for 0.5-2 hours.
4. The method according to claim 1, wherein the mixed enzyme solution is added in the step (4) for digestion for 1 hour.
5. The method according to claim 1, wherein the centrifugation in step (6) is performed at 1000 to 2000rpm for 3 to 7 min.
6. The method according to claim 1, wherein in step (6) the centrifugation is at 1500rpm for 5 min.
7. The method according to claim 1, wherein the seeding density in step (7) is 0.5-2 x105/cm2。
8. The method according to claim 1, wherein the seeding density in step (7) is 1x105/cm2。
9. The method according to claim 1, wherein in step (7), the complete medium has a composition of: DMEM-F12+15% FBS +10ng/ml basic fibroblast growth factor.
10. According to the claimsThe method of claim 1, wherein in the step (7), the culturing is performed at 37 ℃ and 5% CO2Culturing in an incubator.
11. The method of claim 1, wherein the mixed enzyme solution further comprises 0.03% sodium glutamate and 0.075% sodium alginate.
12. 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 a tissue digesting enzyme, said digestive enzyme consisting of: 0.1-0.3% of collagenase II, 0.1-0.2% of collagenase IV and 0.05-0.15% of deoxyribonuclease I; the digestive enzyme composition also comprises 0.02-0.05% of sodium glutamate and 0.05-0.1% of sodium alginate.
13. The digestive enzyme composition according to claim 12, said digestive enzyme consisting of three of: 0.2% collagenase II, 0.15% collagenase IV, 0.1% DNase I.
14. The digestive enzyme composition according to claim 12 further comprising 0.03% sodium glutamate and 0.075% sodium alginate.
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| CN109628388B (en) * | 2018-12-04 | 2023-01-13 | 博雅干细胞科技有限公司 | Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition |
| CN109628393A (en) * | 2019-01-22 | 2019-04-16 | 汇麟生物科技(北京)有限公司 | A kind of method and its digestive juice for separating funicle mesenchyme stem cell |
| CN112662617A (en) * | 2019-10-16 | 2021-04-16 | 中晶生物技术股份有限公司 | Separation and cryopreservation of original point cells and application method |
| CN112458047B (en) * | 2020-11-30 | 2022-09-06 | 博雅干细胞科技有限公司 | Method for separating placenta mesenchymal stem cells and serum-free culture medium used by same |
| CN112608893B (en) * | 2020-12-25 | 2023-03-21 | 博雅干细胞科技有限公司 | Separation and purification method of placenta mesenchymal stem cells |
| CN113181219A (en) * | 2021-03-04 | 2021-07-30 | 纳美细胞科技有限公司 | Cell preparation for removing wrinkles and preparation method thereof |
| CN114369569B (en) * | 2022-01-10 | 2024-04-12 | 中国科学技术大学 | Separation method of umbilical cord Walton gum mesenchymal stem cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630140A (en) * | 2013-11-06 | 2015-05-20 | 吉林济惠生物科技有限公司 | Isolation and culture method of placenta mesenchyma precursor stem cells |
| CN106834257A (en) * | 2017-03-23 | 2017-06-13 | 黑龙江天晴干细胞股份有限公司 | A kind of isolation of human placenta mesenchymal stem mixed enzyme and its separation method |
| CN107236704A (en) * | 2017-06-15 | 2017-10-10 | 博雅干细胞科技有限公司 | From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used |
| WO2017179918A1 (en) * | 2016-04-12 | 2017-10-19 | (주)안트로젠 | Mesenchymal stem cell-hydrogel-biodegradable or mesenchymal stem cell-hydrogel-nondegradable support composition for alleviating or remedying epidermolysis bullosa |
-
2018
- 2018-09-27 CN CN201811130277.0A patent/CN109234229B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630140A (en) * | 2013-11-06 | 2015-05-20 | 吉林济惠生物科技有限公司 | Isolation and culture method of placenta mesenchyma precursor stem cells |
| WO2017179918A1 (en) * | 2016-04-12 | 2017-10-19 | (주)안트로젠 | Mesenchymal stem cell-hydrogel-biodegradable or mesenchymal stem cell-hydrogel-nondegradable support composition for alleviating or remedying epidermolysis bullosa |
| CN106834257A (en) * | 2017-03-23 | 2017-06-13 | 黑龙江天晴干细胞股份有限公司 | A kind of isolation of human placenta mesenchymal stem mixed enzyme and its separation method |
| CN107236704A (en) * | 2017-06-15 | 2017-10-10 | 博雅干细胞科技有限公司 | From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used |
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