CN107299082B - Method for separating placenta mesenchymal cells from tissues and culturing into mesenchymal stem cells - Google Patents
Method for separating placenta mesenchymal cells from tissues and culturing into mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a method for separating placenta mesenchymal cells from tissues and culturing the placenta mesenchymal cells into mesenchymal stem cells, and also relates to mixed enzyme digestive fluid used in the method, in particular to the method which comprises the following steps of treatment of placenta lobules, digestion and termination of mixed enzyme, collection of primary cells, cryopreservation of the primary cells, cell recovery, cell passage and the like7The yield is stable, the P1-P5 generation cells of the mesenchymal stem cells obtained by subculture are mesenchymal stem cells, positive expression CD73, CD90 and CD105 are more than 98 percent, negative expression CD34, CD45, CD19, CD11b and HLA-DR are less than 2 percent; the G2 stage cells of the P5 generation cells are less than 1 percent, have strong proliferation capacity and do not enter the division stage; the ability to differentiate into osteoblasts, adipoblasts and chondroblasts under the stimulation of a specific induction medium.
Description
Technical Field
The present invention relates to a method for isolating stem cells from placenta, particularly to a method for isolating mesenchymal stem cells from placenta, and more particularly to a method for isolating mesenchymal cells from placenta tissue and culturing into mesenchymal stem cells 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 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.
There remains a need in the art for new methods for isolating stem cells from placenta, and in particular for methods that efficiently isolate mesenchymal stem cells from placenta. In addition, there remains a need in the art for new digestive enzyme compositions for use in methods of isolating mesenchymal stem cells from placenta in an effort to improve the efficiency of the methods of isolating mesenchymal stem cells from placenta.
Disclosure of Invention
The invention aims to overcome the defects of the existing method for obtaining the placenta mesenchymal stem cells, and provides a practical, simple and efficient method for separating the mesenchymal cells from the placenta tissues and culturing the mesenchymal stem cells into the placenta mesenchymal stem cells and optionally establishing a placenta stem cell bank. Meanwhile, another object of the present invention is to provide a digestive enzyme composition for the above method for isolating mesenchymal cells from placental tissue and culturing the mesenchymal stem cells. 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 for isolating mesenchymal cells from placental tissue and culturing the mesenchymal cells into mesenchymal stem cells, the method comprising the steps of:
(1) treatment of placental leaflets: placing placenta in a white porcelain dish, and washing with tissue cleaning solutionRemoving blood stasis of placenta, cutting 20g of placenta lobule tissue into a steel cup, cleaning twice with a tissue cleaning solution, soaking for 5min, and weighing 15g of better tissue into a 100mm glass dish; adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm3About the size, 100ml of tissue cleaning fluid is added, the mixture is stirred evenly and filtered by a 300-mesh filter screen, and the operation is repeated to clean the tissue cleaning fluid twice so as to remove blood cells;
[ wherein the tissue cleansing solution is 0.9% physiological saline containing 1% double antibody ]
(2) Mixed enzyme digestion and termination: adding the cleaned leaflet tissue into 15-30 ml (such as 20-25 ml, such as 23ml) of mixed enzyme digestive juice preheated at 37 ℃, fully mixing uniformly, then digesting for 30min by shaking at the temperature of 37 ℃ and 100rpm by using a shaking table, and after digestion is finished, adding 2ml of FBS into the tissue juice to stop digestion;
[ wherein the mixed enzyme digestive juice contains: 15-30 volumes of Hank ' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme, 0.2-2 volumes of DNA type I enzyme (e.g., 20-25 volumes of Hank ' S balanced salt solution, 0.3-0.5 volumes of Liberase MNP-S enzyme, 0.5-1 volumes of DNA type I enzyme, e.g., 22 volumes of Hank ' S balanced salt solution, 0.4 volumes of Liberase MNP-S enzyme, 0.7 volumes of DNA type I enzyme); the Liberase MNP-S enzyme is for example the Liberase MNP-S enzyme from Roche, for example from Siberian organisms, the cat # of which: 5578582001]
(3) Collecting primary cells: adding 50ml of tissue cleaning fluid into the tissue fluid obtained in the last step, uniformly mixing, filtering by a 300-mesh sieve, and collecting cell fluid; washing the digested tissue twice repeatedly, combining the filtrates of the two times into a centrifuge tube, and centrifuging at 1500rpm for 8min (acceleration 9 and deceleration 7); removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, centrifuging at 1500rpm for 8min (acceleration 9, deceleration 7); removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells;
[ cell suspension of this primary cell can be subjected to cell counting using a sysmex hematology analyzer ]
(4) Freezing and storing primary cells: centrifuging the cell suspension at 1800rpm for 10min (acceleration 9 and deceleration 7), collecting cell precipitate and supernatant 5ml, re-suspending, slowly adding frozen stock solution 10ml, and shaking; subpackaging the obtained cell suspension into 9 freezing tubes of 2ml, each tube of 1.5ml, placing in a precooled program cooling box, performing program cooling by using a program cooling instrument, and transferring the cells into a liquid nitrogen storage tank for freezing;
[ wherein, the formula of the freezing solution is as follows: 65% DMEM-F12, 15% Human Serum Albumin (HSA), 20% DMSO, e.g. WAK brand DMSO ]
(5) Cell recovery: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; centrifuging at 1200rpm for 5min (acceleration 9, deceleration 7), removing supernatant, and adding 5ml complete culture medium for resuspension; inoculating each tube of cells into 1T 75 culture bottle, supplementing complete culture medium to 30ml, and culturing in CO2 incubator (37 deg.C, 5% CO2, saturated humidity); performing total liquid change every 3-4 days with complete culture medium, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm2The following passages can be performed;
[ wherein, the complete medium is DMEM-F12 medium containing 10% FBS ]
(6) Cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of the cells fall off, adding 5ml complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9, deceleration 7), discarding supernatant, adding 5ml complete culture medium for resuspension, counting, and inoculating to a culture bottle, wherein the cell density is 8000-12000 cells/cm2Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation; repeating the operations in sequence to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain the mesenchymal stem cells of each generation.
The method according to any embodiment of the first aspect of the present invention, further comprising:
(7) aiming at the placenta mesenchymal stem cells obtained in the step (6), detecting at least one item of the following items: cell viability, cell contamination, genetic disease, HLA-ABC/DR match.
The method according to any embodiment of the first aspect of the present invention, further comprising:
(8) and (4) freezing and storing the passage-generated placenta mesenchymal stem cells obtained in the step (6) in liquid nitrogen.
The method according to any embodiment of the first aspect of the present invention, further comprising:
(9) establishing a database of placental stem cells comprising the above information, and correlating the database with the cryopreserved cells of step (8).
The method according to any one of the embodiments of the first aspect of the present invention, wherein the cell purity of the obtained generations of placental mesenchymal stem cells is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.
The method according to any embodiment of the first aspect of the present invention wherein the Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl 2& 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. The method according to any one of the embodiments of the first aspect of the present invention, wherein the mixed enzyme digestive juice further comprises 0.2-0.3 g/L zinc chloride in addition to Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA type I enzyme. It has been surprisingly found that with the use of mixed enzyme digests with added zinc chloride in this concentration range, the resulting primary cells have greater than 60% CD73 expression, no CD45 expression, and mesenchymal stem cell content in the resulting primary cells of 60% -70%, showing extremely high stem cell concentrations; when the zinc chloride is not added into the mixed enzyme digestive juice, the content of the mesenchymal stem cells in the primary cells is less than 38 percent, and is usually within the range of 31-38 percent.
The method according to any one of the embodiments of the first aspect of the present invention, wherein said cell activity detection is counting the number of viable cells before and after cryopreservation using trypan blue staining.
The method according to any one of the embodiments of the first aspect of the present invention, wherein the cell contamination detection detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B, hepatitis C, HIV, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.
The method according to any one of the embodiments of the first aspect of the present invention, wherein said detection of genetic disease is a method of detecting the presence of genetic disease in cryopreserved cells using molecular genetics.
The method according to any one of the embodiments of the first aspect of the invention, wherein said HLA-ABC/DR matching is the detection of the HLA-ABC/DR phenotype of the cell.
The method according to any one of the embodiments of the first aspect of the present invention, wherein said placental mesenchymal stem cells are frozen in liquid nitrogen via a temperature-programmed process.
A method according to any embodiment of the first aspect of the invention, wherein the database comprises 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, there is provided a placental mesenchymal stem cell. Thus in a second aspect the invention provides a placental mesenchymal stem cell.
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, wherein the cell purity of each generation of placental mesenchymal stem cells is greater than 90%. In one embodiment, the placental mesenchymal stem cells have a cell purity of greater than 95% after more than 3 passages.
Further, the third aspect of the present invention provides a mixed enzyme digest used in a method for separating mesenchymal cells from placental tissue and culturing the mesenchymal stem cells, the mixed enzyme digest comprising Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA type I enzyme.
The mixed enzyme digestive juice according to any embodiment of the third aspect of the present invention comprises: 15-30 volumes of Hank ' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme, 0.2-2 volumes of DNA type I enzyme (e.g., 20-25 volumes of Hank ' S balanced salt solution, 0.3-0.5 volumes of Liberase MNP-S enzyme, 0.5-1 volumes of DNA type I enzyme, e.g., 22 volumes of Hank ' S balanced salt solution, 0.4 volumes of Liberase MNP-S enzyme, 0.7 volumes of DNA type I enzyme).
The mixed enzyme digest according to any one of the embodiments of the third aspect of the present invention, wherein the Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl 2& 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4. The mixed enzyme digest according to any one of the embodiments of the third aspect of the present invention, wherein zinc chloride is added in a prescribed amount as described herein in addition to Hank' S balanced salt solution, Liberase MNP-S enzyme, DNA type I enzyme. It has been surprisingly found that superior technical effects as described in the present invention can be exhibited in the case of using a mixed enzyme digest to which zinc chloride is added in this concentration range.
The mixed enzyme digestion solution according to any embodiment of the third aspect of the present invention, wherein the method for separating mesenchymal cells from placental tissue and culturing into mesenchymal stem cells, comprises the steps of:
(1) treatment of placental leaflets: placing placenta in a white porcelain dish, washing with tissue cleaning solution to remove blood stasis of placenta, shearing 20g of placenta lobule tissue in a steel cup, cleaning twice with the tissue cleaning solution, soaking for 5min, and weighing 15g of better tissue in a 100mm glass dish; adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm3About the left and right size, adding 100ml of tissue cleaning fluid, stirring uniformly, filtering with a 300-mesh filter screen, and filteringThis operation was repeated to wash twice with a tissue wash to remove blood cells;
[ wherein the tissue cleansing solution is 0.9% physiological saline containing 1% double antibody ]
(2) Mixed enzyme digestion and termination: adding the cleaned leaflet tissue into 15-30 ml (such as 20-25 ml, such as 23ml) of mixed enzyme digestive juice preheated at 37 ℃, fully mixing uniformly, then digesting for 30min by shaking at the temperature of 37 ℃ and 100rpm by using a shaking table, and after digestion is finished, adding 2ml of FBS into the tissue juice to stop digestion;
[ wherein the mixed enzyme digestive juice contains: 15-30 volumes of Hank ' S balanced salt solution, 0.2-0.6 volumes of Liberase MNP-S enzyme, 0.2-2 volumes of DNA type I enzyme (e.g., 20-25 volumes of Hank ' S balanced salt solution, 0.3-0.5 volumes of Liberase MNP-S enzyme, 0.5-1 volumes of DNA type I enzyme, e.g., 22 volumes of Hank ' S balanced salt solution, 0.4 volumes of Liberase MNP-S enzyme, 0.7 volumes of DNA type I enzyme); the Liberase MNP-S enzyme is for example the Liberase MNP-S enzyme from Roche, for example from Siberian organisms, the cat # of which: 5578582001]
(3) Collecting primary cells: adding 50ml of tissue cleaning fluid into the tissue fluid obtained in the last step, uniformly mixing, filtering by a 300-mesh sieve, and collecting cell fluid; washing the digested tissue twice repeatedly, combining the filtrates of the two times into a centrifuge tube, and centrifuging at 1500rpm for 8min (acceleration 9 and deceleration 7); removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, centrifuging at 1500rpm for 8min (acceleration 9, deceleration 7); removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells;
[ cell suspension of this primary cell can be subjected to cell counting using a sysmex hematology analyzer ]
(4) Freezing and storing primary cells: centrifuging the cell suspension at 1800rpm for 10min (acceleration 9 and deceleration 7), collecting cell precipitate and supernatant 5ml, re-suspending, slowly adding frozen stock solution 10ml, and shaking; subpackaging the obtained cell suspension into 9 freezing tubes of 2ml, each tube of 1.5ml, placing in a precooled program cooling box, performing program cooling by using a program cooling instrument, and transferring the cells into a liquid nitrogen storage tank for freezing;
[ wherein, the formula of the freezing solution is as follows: 65% DMEM-F12, 15% Human Serum Albumin (HSA), 20% DMSO, e.g. WAK brand DMSO ]
(5) Cell recovery: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; centrifuging at 1200rpm for 5min (acceleration 9, deceleration 7), removing supernatant, and adding 5ml complete culture medium for resuspension; inoculating each tube of cells into 1T 75 culture bottle, supplementing complete culture medium to 30ml, and culturing in CO2 incubator (37 deg.C, 5% CO2, saturated humidity); performing total liquid change every 3-4 days with complete culture medium, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm2The following passages can be performed;
[ wherein, the complete medium is DMEM-F12 medium containing 10% FBS ]
(6) Cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of the cells fall off, adding 5ml complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9, deceleration 7), discarding supernatant, adding 5ml complete culture medium for resuspension, counting, and inoculating to a culture bottle, wherein the cell density is 8000-12000 cells/cm2Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation; repeating the operations in sequence to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain the mesenchymal stem cells of each generation.
The mixed enzyme digestion solution according to any embodiment of the third aspect of the present invention, wherein the method for separating mesenchymal cells from placenta tissue and culturing into mesenchymal stem cells further comprises:
(7) aiming at the placenta mesenchymal stem cells obtained in the step (6), detecting at least one item of the following items: cell viability, cell contamination, genetic disease, HLA-ABC/DR match.
The mixed enzyme digestion solution according to any embodiment of the third aspect of the present invention, wherein the method for separating mesenchymal cells from placenta tissue and culturing into mesenchymal stem cells further comprises:
(8) and (4) freezing and storing the passage-generated placenta mesenchymal stem cells obtained in the step (6) in liquid nitrogen.
The mixed enzyme digestion solution according to any embodiment of the third aspect of the present invention, wherein the method for separating mesenchymal cells from placenta tissue and culturing into mesenchymal stem cells further comprises:
(9) establishing a database of placental stem cells comprising the above information, and correlating the database with the cryopreserved cells of step (8).
The mixed enzyme digestion solution according to any embodiment of the third aspect of the present invention, wherein the cell activity detection in the method of isolating mesenchymal cells from placenta tissue and culturing into mesenchymal stem cells is counting the number of viable cells before and after cryopreservation using trypan blue staining method.
The mixed enzyme digestion solution according to any embodiment of the third aspect of the present invention, wherein the cell contamination detection in the method of separating mesenchymal cells from placenta tissue and culturing into mesenchymal stem cells detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture. In one embodiment, the cell contamination assay utilizes an etiological method to detect whether a cell is infected with one or more of the following: hepatitis B, hepatitis C, HIV, cytomegalovirus, EB virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, and TRUST.
The mixed enzyme digestion solution according to any embodiment of the third aspect of the present invention, wherein the genetic disease detection in the method of isolating mesenchymal cells from placental tissue and culturing into mesenchymal stem cells is a method of detecting the presence or absence of a genetic disease in cryopreserved cells using a molecular genetic method.
The mixed enzyme digest of any embodiment of the third aspect of the present invention, wherein said HLA-ABC/DR typing in said method of isolating mesenchymal cells from placental tissue and culturing into mesenchymal stem cells is detecting the HLA-ABC/DR phenotype of the cells.
The mixed enzyme digestion solution according to any embodiment of the third aspect of the present invention, wherein the method for separating mesenchymal cells from placental tissue and culturing into mesenchymal stem cells comprises freezing the mesenchymal stem cells in liquid nitrogen through a temperature programming process.
The mixed enzyme digestion solution according to any embodiment of the third aspect of the present invention, wherein the method for separating mesenchymal cells from placental tissue and culturing into mesenchymal stem cells comprises in the database 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.
Of the various process steps described above, although specific steps are described in some detail or in language specific to the process steps described in the examples of the following detailed description, those skilled in the art will be able to fully appreciate the above-described process steps from the detailed disclosure of the invention as a whole.
Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
In the present invention, the term "placental mesenchymal stem cells" refers to mesenchymal stem cells derived from the placenta. Thus in the present invention, and in particular in the context relating to the present invention, the term "placental mesenchymal stem cells" may be used interchangeably with "placental stem cells", "mesenchymal stem cells", unless otherwise specifically indicated.
In the present invention, the term "PBS buffer" or "PBS" refers to a phosphate buffer. The general formulation and formulation of the PBS used in the context of the present invention, as well as their general properties such as pH value or pH range, are well known to those skilled in the art and are typically commercially available pre-formulations (or powders), e.g. the PBS used in the field of the present invention is typically a commercial buffer at pH7.4(± 0.1), e.g. HyClone brand PBS buffer; in the present invention, the composition of PBS buffer solution in the classical application of the art includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate, and PBS used in the present invention has the same composition as that in the present invention, unless otherwise specified.
In the present invention, the term "placenta" refers to the placenta of a newborn, and in particular to the placenta within 4 hours of birth.
The inventor of the invention utilizes a perfusion method to separate and culture the mesenchymal stem cells from the placenta, and obtains the mesenchymal stem cells with high purity. However, after perfusion, a large amount of stem cells still remain in the placenta tissue and cannot be effectively separated. Therefore, it is considered that the mesenchymal stem cells cannot be obtained to the maximum extent by the perfusion method.
The invention discloses a method for separating a large number of mesenchymal stem cells from placenta, and the method is used for preserving the mesenchymal stem cells of the placenta and establishing a placenta stem cell bank. Based on the summary of the conventional separation and culture of the mesenchymal stem cells, the inventor successfully separates and obtains a large amount of mesenchymal stem cells from the placenta by utilizing a mixed digestion of a plurality of tissue digestive enzymes and combining an adherence culture method. The mesenchymal stem cells obtained by the method have high purity and large quantity, have the same biological characteristics as the mesenchymal stem cells of the bone marrow, and can be differentiated into osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells and the like. Because stem cells in the placenta are more immature than adult stem cells and rich in content, the placenta stem cells have wide application prospects in clinic, the mesenchymal stem cells are cryopreserved like cord blood by using a conventional cell cryopreservation method, a placenta stem cell bank is established, and a foundation is laid for the further research and clinical treatment of the stem cells.
Because the cord blood contains abundant hematopoietic stem cells, people establish a cord blood bank to store the cord blood hematopoietic stem cells which are an important biological resource, and a treatment means is provided for various blood system diseases and immune system diseases. Similarly, the placenta mesenchymal stem cells are used as a more important stem cell resource, and are frozen in liquid nitrogen at the deep low temperature of-196 ℃ for long-term storage by using a conventional cell freezing storage method, so that a placenta stem cell bank is established, and seeds are preserved for the treatment of stem cells in the future.
Particularly, the method can obtain primary cells with extremely high mesenchymal stem cell purity in the P0 generation, the expression of CD73 of the primary cells is more than 60 percent, the expression of CD45 of the primary cells is not carried out, and the content of the mesenchymal stem cells in the primary cells reaches 60 to 70 percent.
For example, the invention selects a full-term placenta sample, cuts 15g of tissue at a specific position of a placenta lobule, digests the tissue by a mixed enzyme system, obtains cells, and then purifies the cells to obtain a group of relatively pure mesenchymal cells (the expression of CD73 is more than 60 percent, the expression of CD45 is not more than 32 percent), and the number of the obtained cells per gram of the tissue can reach 2.5 × 107And the yield is stable, and the sample specificity is greatly reduced. The batch of primary interstitial cells are subjected to recovery culture after being frozen, and are cultured by using a classical complete culture medium formula, so that more spindle-type adherent cells can be microscopically detected in about 4 days, the cell fusion rate reaches 70-80% in 10 days, and the cells can be transferred to P1 generations. After continuous passage to P5 generation, carrying out experiments such as flow type phenotype identification, cell cycle detection, induced differentiation and the like, wherein the results show that the cells of P1-P5 generation are mesenchymal stem cells, the positive expression (CD73, CD90 and CD105) is more than 98 percent, and the negative expression (CD34, CD45, CD19, CD11b and HLA-DR) is less than 2 percent; the G2 stage cells of the P5 generation cells are less than 1 percent, have strong proliferation capacity and do not enter the division stage; under the stimulation of a specific induction culture medium,and the ability to differentiate into osteoblasts, adipoblasts and chondroblasts.
The method is simple to operate, convenient and practical, can obtain a large amount of mesenchymal stem cells, has good differentiation performance, and has the capacity of differentiating osteoblasts, adipocytes, chondrocytes, endothelial cells, nerve cells and other cells. Comparison with existing methods: at present, MSC is mainly obtained by extracting donor bone marrow by an operation method or separating placenta by a perfusion method and carrying out adherent culture. The method has the advantages of low cell number, and infection possibility in marrow extraction and after marrow extraction. The invention successfully separates and obtains a large amount of mesenchymal stem cells with higher purity from the placenta, and establishes a placental stem cell bank by using the method to store the stem cells with great application prospect. The method is simple and easy to implement, and because the placenta is the same as cord blood, the cell components are more immature, the source is wide, and the method is convenient and easy to obtain, the method has wide prospect in the clinical application of stem cells.
Drawings
FIG. 1: the flow type phenotype identification result of the primary cell obtained by the invention is shown in the figure.
FIG. 2: micrographs of samples during passage P0.
FIG. 3: micrographs of samples during passage P5.
FIG. 4: the samples were phenotyped at P5.
FIG. 5: DNA content-cell number relationship of the sample P5 generation cells.
FIG. 6: the induced differentiation test of the P5 generation cells shows that the cells have the capacity of differentiating to osteogenic, adipogenic and chondrogenic cells.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
Example 1, placental whole cell treatment:
1. preparing a mixed synthase digestive solution: 22ml of HBSS (Hank' S balanced salt solution) containing calcium and magnesium ions, 0.4ml of Liberase Roche MNP-S enzyme (purchased from Sibao, for example, cat # 5578582001) and 0.7ml of DNA I enzyme were transferred into a 50ml centrifuge tube, and zinc chloride (added at a concentration of 0.2g/L, 0.25g/L or 0.3g/L) was added thereto, mixed, and preheated at 37 ℃ for 20min or more. The Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4 & 7H2O, 0.1g/L MgCl 2& 6H2O, 0.06g/L Na2HPO4 & 2H2O, 0.06g/L KH2PO4, 1.0g/L glucose, 0.14g/L CaCl2, 0.35 g/L3, 0.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4.
2. Preparation of placental leaflets: the placenta is taken out from the collection bag and placed in a white porcelain dish, the blood stasis of the placenta is removed after the tissue cleaning solution is washed, and a small amount of placenta lobule tissue (about 20 g) is cut and placed in a steel cup. The tissue is washed twice with tissue washing solution (0.9% physiological saline + double antibody (double antibody is streptomycin, content 1%)), and after soaking for 5min, 15g + -1 g of better tissue is weighed into a 100mm glass dish.
3. Removal of blood cells: adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm3About the size, 100ml of tissue cleaning fluid is added and stirred evenly, then the mixture is filtered by a 300-mesh filter screen, and then the mixture is cleaned twice by the tissue cleaning fluid (the leaflet tissue is moved into a steel cup each time, 100ml of tissue cleaning fluid is added and stirred evenly, and then the mixture is filtered by a 300-mesh filter screen).
4. Mixed enzyme digestion and termination: adding the cleaned leaflet tissue into preheated 23ml of mixed enzyme digestive juice, fully and uniformly mixing, and then oscillating and digesting for 30min by a shaking table at 37 ℃ and 100 rpm. After digestion, the tissue fluid +2ml FBS was terminated.
5. Collecting primary cells:
adding 50ml of tissue cleaning fluid to dilute and mix the tissue fluid uniformly, filtering by a 300-mesh sieve, collecting cell fluid, washing the digested tissue twice (50 ml of tissue cleaning fluid is used each time), merging the filtrate into 1 250ml of centrifuge tubes, and centrifuging at 1500rpm for 8min (acceleration 9 and deceleration 7);
removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, centrifuging at 1500rpm for 8min (acceleration 9, deceleration 7);
removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells; 1ml of suspension is taken and used for a sysmex hematology analyzer to count cells, and the primary cells are high in purity and have the mesenchymal stem cell content of about 60-70 percent through determination.
6. Freezing and storing primary cells:
the frozen stock solution is prepared on site, and the formula of the frozen stock solution is as follows: 65% DMEM-F12, 15% Human Serum Albumin (HSA), 20% DMSO, e.g., WAK brand DMSO, ready for use;
centrifuging the cell suspension at 1800rpm for 10min (acceleration 9 and deceleration 7), collecting cell precipitate and supernatant 5ml (sample of supernatant 10 ml), re-suspending, slowly adding the prepared frozen stock solution while shaking;
the cell suspension was dispensed into 9 2ml cryopreserved tubes, 1.5ml per tube (pre-cooled in a programmed cooling box). The remaining cell suspension + the retained sample supernatant are used for sterile detection;
and (4) performing program cooling by using a program cooling instrument, transferring the cells into a liquid nitrogen storage tank, and performing cryopreservation on the obtained primary cells.
Through the whole placenta cell processing process of the embodiment 1, a placenta sample of full term is selected, 15g of tissue at a specific position of a placenta lobule is cut, the tissue is digested by the mixed enzyme digestive fluid system, cells are obtained and then purified, and a group of relatively pure primary mesenchymal cells (the expression of CD73 is more than 60 percent, and the expression of CD45 is not carried out) can be obtained, wherein the content of mesenchymal stem cells in the primary mesenchymal cells reaches 60-70 percent, and the number of the primary cells obtained from each gram of the placenta lobule tissue can reach (2.4-2.8) × 107However, when the zinc chloride is not added into the mixed enzyme digestive fluid, the content of mesenchymal stem cells in primary cells is less than 38 percent, usually in the range of 31-38 percent, and the number of the primary cells obtained per gram of the placenta lobule tissue is less than 5 × 105A plurality of; in addition, the present inventionThe inventors found that primary cell numbers obtained per gram of placental leaflet tissue were less than 2 × 10 when prepared by reference to other prior art primary cell preparations6And 1/10 or less in the method of the present invention. The whole cell treatment of the placenta can efficiently obtain primary mesenchymal cells, and lays a good foundation for the subsequent culture of mesenchymal stem cells with high medical value.
For example, in this example, the cell yield was very stable after placental tissue treatment, and typical data for some experiments are shown in table 1.
Table 1: cell yield of primary cells from placental tissue
| Date of treatment | Sample registration number | Tissue volume (g) | Cell number (× 10)8) | Cell yield (10)8/g) |
| 2016.7.22 | 9004116082279 | 15 | 3.8 | 0.25 |
| 2016.7.25 | 9004116082301 | 15.9 | 3.9 | 0.25 |
| 2016.7.26 | 9004116082311 | 14.9 | 3.8 | 0.26 |
| 2016.7.27 | 9004116082319 | 14.9 | 3.8 | 0.26 |
| 2016.8.25 | 9004116082539 | 15 | 3.75 | 0.25 |
In addition, the flow phenotype identification result of the primary cells obtained after the placenta treatment shows that the expression of CD73 is up to more than 60%, and CD45 is not expressed, which indicates that the primary cells are a group of relatively pure mesenchymal cells and do not contain blood cells. The flow phenotyping results are shown in FIG. 1.
Example 2 recovery and subculture of Primary cells
1. Cell recovery:
taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; as not otherwise specified, complete medium as used herein is DMEM-F12 medium containing 10% FBS;
centrifuging at 1200rpm for 5min (acceleration 9, deceleration 7), removing supernatant, and adding 5ml complete culture medium for resuspension; inoculating each tube of cells into 1T 75 culture bottle, supplementing complete culture medium to 30ml, and culturing in CO2 incubator (37 deg.C, 5% CO2, saturated humidity); completely culturing every 3-4 daysPerforming primary total fluid exchange, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm2The following passages can be performed;
2. cell passage: washing recovered P0 generation cells with PBS, adding 2ml of pancreatin for digestion for 2-5min until most of the cells fall off, adding 5ml of complete culture medium to stop digestion, transferring the cells into a centrifuge tube, centrifuging at 1400rpm for 5min (acceleration 9, deceleration 7), discarding supernatant, adding 5ml of complete culture medium for resuspension, counting, and inoculating to a culture bottle, wherein the cell density is 8000-12000 cells/cm2Culturing in a CO2 incubator (37 ℃, 5% CO2 and saturated humidity) until the cell density reaches over 90% (usually culturing for about 5 days), and completing cell passage from P0 generation to P1 generation;
the passage operations from the generation P0 to the generation P1 are sequentially repeated to perform cell passages from the generation P1 to the generation P2, from the generation P2 to the generation P3, from the generation P3 to the generation P4 and from the generation P4 to the generation P5 respectively, so that the mesenchymal stem cells of each generation are obtained.
In the present embodimentThe primary seeding density is about 5 × 105cells/cm2And a plurality of adherent cells are arranged in the field of microscopic examination for 4 days after inoculation and are spindle-shaped. The strain can be transferred to P1 generation 10 days after inoculation. The cells grow fast and are full, and the cells are in spindle shapes. During passage from P0 to P1, the cell counts for some experiments are shown schematically in table 2 below. The micrograph of the PS162279 sample during passaging is shown in FIG. 2.
Table 2: cell count results during passage from passage P0 to passage P1
| Sample registration number | P0-P1 | P0 count |
| PS162311 | D10 | 8*105(ADAM) |
| PS162319 | D10 | 1.4*106(ADAM) |
| PS162279 | D9 | 1.5*106(ADAM) |
In addition, in the process of inoculating and passaging P1-P5, the seeds are usually cultured for 4-5 days, harvested and passaged to the next generation. An exemplary micrograph of the PS162279 sample during passage P5 is shown in figure 3.
In this experiment, the flow phenotype identification of P1-P5 generation was carried out, the results showed that the positive expression of CD73, CD90 and CD105 was > 98%, and CD34, CD45, CD19 and HLA-DR were identified, and the results are shown in Table 3, which proved that the cells isolated and cultured in placenta were mesenchymal stem cells and had high purity.
Table 3: flow phenotyping results of P1-P5 generation cells
Exemplary, the P5 representative flow phenotypic identification results of the PS162279 sample are shown in fig. 4.
In addition, the growth cycle of some samples was measured for P5 generation cells, and the results showed < 1% G2 phase cells and > 10% S phase cells, demonstrating that these cells are highly proliferative and do not enter the division phase, and the specific results are shown in Table 4.
Table 4: growth cycle assay results for P5 generation cells
| Sample registration number | Stage GO/G1 | Stage S | Stage G2/M |
| PS162311-P5 | 84.60％ | 14.80％ | 0.64％ |
| PS162319-P5 | 82.30％ | 16.80％ | 0.93％ |
| PS162279-P5 | 87.00％ | 11.90％ | 0.80％ |
In addition, the DNA content-cell number relationship was plotted for PS162311-P5 cells, and typical results are shown in FIG. 5
Example 3 biological characterization of placental MSCs
Reference to issued patents0062 to CN102676451A]To [0089]The method for identifying the biological characteristics of the placenta mesenchymal stem cells shows that the MSC separated by the method has the capacity of differentiating osteoblasts, adipocytes and chondrocytes, and the MSC obtained by the method has the characteristics of the stem cells.
For example, the P5 generation cells were illustratively tested for induced differentiation and showed the ability to differentiate into osteogenic, adipogenic and chondrogenic cells. A typical micrograph of adipogenic, osteogenic, and chondrogenic differentiation is shown in fig. 6.
Example 4 establishment of placental stem cell Bank
1. Detection of cellular Activity
The number of viable cells before and after cryopreservation was counted using trypan blue staining.
2. Detection of cell contamination
And (3) detecting whether the cells are polluted by fungi and bacteria by using a small amount of cell culture. And (3) detecting whether the cells are infected by hepatitis B two-and-two, hepatitis C, AIDS, cytomegalovirus, EB virus, syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM, EBV-IgA and TRUST by utilizing an etiological method.
3. Detection of genetic disorders
And detecting whether the frozen cells have genetic diseases or not by using a molecular genetics method.
4. HLA-ABC/DR match
Cells were tested for HLA-ABC/DR phenotype and recorded.
5. Investigation of cell origin
The details of the fetus and its parents are recorded and recorded on the record.
6. Establishment of placental stem cell database
After normal placental stem cells are preserved, a database of placental stem cells is established, which includes the first six data, and associations with cryopreserved cells are established.
Claims (11)
1. A method for isolating mesenchymal cells from placental tissue and culturing into mesenchymal stem cells, the method comprising the steps of:
(1) treatment of placental leaflets: placing placenta in a white porcelain dish, washing with tissue cleaning solution to remove blood stasis, shearing 20g of placenta lobule tissue in a steel cup, cleaning twice with the tissue cleaning solution, soaking for 5min, and weighing 15g of better tissueIn a 100mm glass dish; adding 10ml tissue cleaning solution, and cutting leaflets to 0.2cm3Adding 100ml of tissue cleaning fluid, stirring uniformly, filtering by a 300-mesh filter screen, and repeating the operation to clean twice by using the tissue cleaning fluid so as to remove blood cells;
(2) mixed enzyme digestion and termination: adding the cleaned leaflet tissue into 15-30 ml of mixed enzyme digestive juice preheated at 37 ℃, fully mixing uniformly, then digesting for 30min by shaking at 37 ℃ and 100rpm in a shaking table, and after digestion is finished, adding 2ml of FBS into the tissue juice to stop digestion;
(3) collecting primary cells: adding 50ml of tissue cleaning fluid into the tissue fluid obtained in the last step, uniformly mixing, filtering by a 300-mesh sieve, and collecting cell fluid; washing the digested tissue twice repeatedly, combining the filtrates of the two times into a centrifuge tube, and centrifuging at 1500rpm for 8 min; removing supernatant, adding appropriate amount of tissue washing solution, resuspending and supplementing to 200ml, and centrifuging at 1500rpm for 8 min; removing supernatant, adding DMEM-F12 into the cell sediment, resuspending the cell sediment to 30ml, filtering with a 100um filter screen, and then washing the filter screen with 10ml DMEM-F12 to obtain 40ml cell suspension as primary cells;
(4) freezing and storing primary cells: centrifuging the cell suspension at 1800rpm for 10min, collecting cell precipitate and lower solution 5ml, re-suspending, slowly adding frozen solution 10ml while shaking; subpackaging the obtained cell suspension into 9 freezing tubes of 2ml, each tube of 1.5ml, placing in a precooled program cooling box, performing program cooling by using a program cooling instrument, and transferring the cells into a liquid nitrogen storage tank for freezing;
(5) cell recovery: taking 2 tubes of frozen cells, quickly thawing at 37 ℃, transferring the cells to a 15ml centrifuge tube, and adding 8ml of complete culture base for resuscitating; centrifuging at 1200rpm for 5min, removing supernatant, and adding 5ml complete culture medium for resuspension; each tube of cells was inoculated into 1T 75 flask, supplemented with complete medium to 30ml, CO was added2Culturing in an incubator; performing total liquid change every 3-4 days with complete culture medium, recovering for 12 days, and counting according to clone formation condition until cell density is not less than 3000 cells/cm2The following passages can be performed;
(6) cell passage: washing P0 generation cells with PBS, adding 2ml pancreatin for 2-5min until most of cells are shed, adding 5ml, and completely culturingStopping digestion of the medium, transferring the cells into a centrifuge tube, centrifuging for 5min at 1400rpm, discarding the supernatant, adding 5ml of complete medium for resuspension, counting and inoculating the cells into a culture flask, wherein the cell density is 8000-12000 cells/cm2Placing CO in2Culturing in an incubator until the cell density reaches over 90 percent, and completing cell passage from P0 generation to P1 generation; sequentially repeating the operations to perform cell passage from P1 generation to P2 generation, P2 generation to P3 generation, P3 generation to P4 generation and P4 generation to P5 generation respectively to obtain mesenchymal stem cells of each generation;
(7) aiming at the placenta mesenchymal stem cells obtained in the step (6), detecting at least one item of the following items: cell viability, cell contamination, genetic disease, HLA-ABC/DR match;
(8) freezing and storing the passage-generated placenta mesenchymal stem cells obtained in the step (6) in liquid nitrogen;
(9) establishing a database of placental stem cells comprising information on cell viability, cell contamination, genetic disease, HLA-ABC/DR match, and correlating the database with the cryopreserved cells of step (8),
the method is characterized in that:
the mixed enzyme digestive juice comprises: 15-30 volumes of Hank' S balanced salt solution, 0.2-0.6 volume of LiberasemNP-S enzyme, 0.2-2 volumes of DNA I-type enzyme and 0.2-0.3 g/L of zinc chloride;
the Hank's balanced salt solution consists of: 8.0g/L NaCl, 0.4g/L KCl, 0.1g/L MgSO4·7H2O, 0.1g/L MgCl2·6H2O, 0.06g/L Na2HPO4·2H2O, 0.06g/L KH2PO41.0g/L glucose, 0.14g/L CaCl20.35g/L NaHCO30.2g/L phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4.
2. The method of claim 1, wherein the tissue wash is 0.9% saline containing 1% double antibody.
3. The method of claim 1, wherein said mixed enzyme digest comprises: 20-25 volumes of Hank' S balanced salt solution, 0.3-0.5 volumes of Liberase MNP-S enzyme and 0.5-1 volumes of DNA I-type enzyme.
4. The method of claim 1, wherein the formulation of the cryopreservation solution is: 65% DMEM-F12, 15% human serum albumin, 20% DMSO.
5. The method according to claim 1, wherein said complete medium is DMEM-F12 medium containing 10% FBS.
6. The method according to claim 1, wherein the cell activity is measured by counting the number of viable cells before and after cryopreservation using trypan blue staining.
7. The method according to claim 1, wherein the cell contamination detection detects whether the cells are contaminated with fungi and bacteria using a small amount of cell culture.
8. The method according to claim 1, wherein the genetic disease detection is a method of molecular genetics for detecting the presence of a genetic disease in cryopreserved cells.
9. The method of claim 1, wherein said HLA-ABC/DR match is a measure of cellular HLA-ABC/DR phenotype.
10. The method of claim 1, wherein said placental mesenchymal stem cells are frozen in liquid nitrogen via a temperature-programmed process.
11. The method according to claim 1, wherein the database includes data relating to all of the cells stored, selected from the group consisting of: 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.
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| CN115777691A (en) * | 2018-04-19 | 2023-03-14 | 重庆斯德姆生物技术有限公司 | Method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells |
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| CN109699634A (en) * | 2019-01-31 | 2019-05-03 | 和携科技(北京)有限公司 | A kind of Cryopreservation and method for resuscitation of mescenchymal stem cell |
| CN110499287B (en) * | 2019-08-30 | 2021-07-23 | 博雅干细胞科技有限公司 | Method for simply preparing placenta mesenchymal stem cell exosome |
| CN113403260A (en) * | 2020-12-09 | 2021-09-17 | 海南优尼科尔生物科技有限公司 | Preparation and culture method of placenta stem cells |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270349A (en) * | 2008-03-20 | 2008-09-24 | 浙江大学 | Method for placenta mesenchyma stem cell separation and in vitro amplify cultivation |
| CN102586184A (en) * | 2011-09-05 | 2012-07-18 | 博雅干细胞科技有限公司 | Method for establishing placental mesenchyme stem cell library |
| CN102660501A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord |
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
| CN102807966A (en) * | 2012-08-16 | 2012-12-05 | 博雅干细胞科技有限公司 | Method for freezing and thawing placental whole cells and separating and expanding stem cells |
| CN105002136A (en) * | 2015-07-31 | 2015-10-28 | 夏凡 | Separation method of human placenta mesenchymal stem cells |
| CN105018421A (en) * | 2015-05-27 | 2015-11-04 | 贵州北科泛特尔生物科技有限公司 | Culturing method for human placental mesenchymal stem cells |
| CN106754674A (en) * | 2016-12-09 | 2017-05-31 | 博雅干细胞科技有限公司 | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion |
-
2017
- 2017-08-02 CN CN201710653583.1A patent/CN107299082B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270349A (en) * | 2008-03-20 | 2008-09-24 | 浙江大学 | Method for placenta mesenchyma stem cell separation and in vitro amplify cultivation |
| CN102586184A (en) * | 2011-09-05 | 2012-07-18 | 博雅干细胞科技有限公司 | Method for establishing placental mesenchyme stem cell library |
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
| CN102660501A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord |
| CN102807966A (en) * | 2012-08-16 | 2012-12-05 | 博雅干细胞科技有限公司 | Method for freezing and thawing placental whole cells and separating and expanding stem cells |
| CN105018421A (en) * | 2015-05-27 | 2015-11-04 | 贵州北科泛特尔生物科技有限公司 | Culturing method for human placental mesenchymal stem cells |
| CN105002136A (en) * | 2015-07-31 | 2015-10-28 | 夏凡 | Separation method of human placenta mesenchymal stem cells |
| CN106754674A (en) * | 2016-12-09 | 2017-05-31 | 博雅干细胞科技有限公司 | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion |
Non-Patent Citations (3)
| Title |
|---|
| Conditions for Collection of Placental Tissue Samples for Culturing of Multipotent Mesenchymal Stromal Cells Culturing of Multipotent Mesenchymal Stromal Cells;N. V. Nizyaeva et al;《Bulletin of Experimental Biology and Medicine》;20170228;第162卷(第4期);第501-506页 * |
| Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery;LeeLankford et al;《Cytotherapy》;20170630;第19卷(第6期);第680-688页 * |
| 胎盘源间充质干细胞分离提取的新方法;刘洋等;《中国组织工程研究》;20150305;第19卷(第10期);1608-1611 * |
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