CN112646775A - Isolated culture method of human umbilical cord mesenchymal stem cells - Google Patents

Isolated culture method of human umbilical cord mesenchymal stem cells Download PDF

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CN112646775A
CN112646775A CN202011606233.8A CN202011606233A CN112646775A CN 112646775 A CN112646775 A CN 112646775A CN 202011606233 A CN202011606233 A CN 202011606233A CN 112646775 A CN112646775 A CN 112646775A
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徐栋花
杨晶涵
杨春娟
褚锦锦
张娇娇
王菁华
孙家梅
张丽丽
李海波
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Abstract

The invention discloses a separation culture method of human umbilical cord mesenchymal stem cells, which comprises the steps of collecting, storing and transporting umbilical cords, separating Wharton's jelly tissues, extracting and culturing cells, carrying out cell passage and preserving seeds for freezing; the separation method adopted by the invention is to remove the umbilical cord adventitia and the blood vessel to obtain pure Wharton's jelly tissue, so that the purity of the separated mesenchymal stem cells is higher and the number of the separated mesenchymal stem cells is more; the method greatly shortens the preparation period of the primary cells by using a two-step enzyme digestion method, has good cell activity, strong proliferation capacity, good differentiation potential and high separation efficiency, and can particularly meet the cell requirements of large-scale experimental research and clinical application.

Description

Isolated culture method of human umbilical cord mesenchymal stem cells
Technical Field
The invention belongs to the field of stem cells and regenerative medicine, relates to a stem cell separation and culture technology, and particularly relates to a method for separating, extracting and culturing mesenchymal stem cells from human umbilical cord.
Background
The mesenchymal stem cells are special stem cells and have strong self-renewal and multidirectional differentiation potentials. Mesenchymal stem cells have gained high attention in various fields of medicine due to their great potential in immune regulation and tissue repair. The mesenchymal stem cells have wide material sources and can be separated from tissues such as bone marrow, synovium, fat, cord blood, umbilical cord and the like. In addition, the cells can be differentiated into various tissue cells such as osteocytes, endothelial cells, chondrocytes, cardiomyocytes, adipocytes and the like through special induction. At present, the main research source of the mesenchymal stem cells is bone marrow, on one hand, the bone marrow acquisition difficulty is high, on the other hand, the proliferation and differentiation activity of the mesenchymal stem cells prepared from the bone marrow are influenced by the age increase and the virus susceptibility exists, so the demand of searching the bone marrow substitute source of the mesenchymal stem cells is increasingly urgent.
Research shows that a large amount of high-purity mesenchymal stem cells can be extracted and obtained from human umbilical cord Wharton's jelly tissues. The umbilical cord has the advantages of rich source, convenient and unlimited material taking, no ethical dispute and the like, and provides a new source way for obtaining the mesenchymal stem cells. Like bone marrow-derived mesenchymal stem cells, umbilical cord-derived cells also have the functional characteristics of low immunogenicity, regulating immunoinflammation, promoting tissue injury repair and the like, and are expected to be widely applied to clinic as an effective treatment strategy.
However, how to efficiently separate and obtain relatively pure mesenchymal stem cells from umbilical cord Wharton's jelly tissue is still a major problem that retards the progress of current research. At present, the mainstream methods for extracting mesenchymal stem cells from umbilical cord Wharton's jelly tissues comprise a protease digestion method and a tissue block adherence method. The conventional protease digestion method mainly uses collagenase, hyaluronidase and neutral protease to obtain Cells, the digestion time is up to 6 hours or more, which not only prolongs the in vitro separation time of the Cells, but also easily causes the influence on the activity of the Cells (BeravoluN, et al, isolation and Characterization of Mesenchyl chemical Cells from Human ultra and Fetal plant A. J. Vis Exp.2017; (122): 55224; Zhang Z, et al, Human ultra and mental Stem Cells inhibition of insulin Lung promoter expression of insulin Lung-protein B6. MRL-lpr Mi NOS.2017; 26(6):1031 Fas.1042). Although the tissue block wall-adhering method has the advantages of simple process and low cost, the cell acquisition period is long, the cell acquisition rate is low, the requirements of large-scale experimental research and clinical application are difficult to meet, and the experimental progress is seriously influenced.
Disclosure of Invention
Aiming at the defects, the invention provides a more efficient umbilical cord mesenchymal stem cell separation culture method, which simplifies the operation process, shortens the enzyme action time, does not need special digestion tools, can greatly shorten the separation period of primary cells, and can quickly obtain a large amount of mesenchymal stem cells with high purity and high activity.
The invention is realized by the following steps:
a method for extracting and culturing mesenchymal stem cells from umbilical cord Wharton's jelly tissue comprises the following steps:
(1) collecting, storing and transporting umbilical cords: obtaining an umbilical cord under an aseptic condition, preliminarily cleaning the internal arterial venous blood of the umbilical cord, ligating two ends of the umbilical cord by using rubber bands, soaking the umbilical cord in aseptic normal saline, storing and transporting at a low temperature (0-4 ℃), and processing within 6 hours;
(2) separating the tissue of the Wharton's jelly: repeatedly washing with sterile normal saline, removing residual blood on the surface of the umbilical cord and in blood vessels, dividing the umbilical cord into segments of about 3-4cm by using sterile scissors, placing the segments of the umbilical cord on sterile gauze soaked by normal saline, longitudinally cutting off umbilical cord adventitial tissues from a shallow layer by using an ophthalmic scissors, fixing one side of the adventitia, scraping and stripping Wharton's jelly and the adventitia by using a scalpel, and then removing the blood vessels by using hemostatic forceps, wherein tissue moistening is ensured in the process;finally, washing the separated Wharton's jelly tissue by using sterile normal saline, and shearing the Wharton's jelly tissue into pieces of 1-3mm3Tissue mass;
(3) cell extraction and culture:
(3.1) adding a collagenase solution to the minced tissue mass for the first digestion, wherein the volume ratio of the collagenase solution to the tissue mass is preferably 1: 1; incubating and digesting at 37 deg.C for 30min, centrifuging at 3000 Xg for 8-10min, discarding supernatant, and collecting precipitate; the collagenase solution is prepared by dissolving 12500U/mL collagenase type II (Sigma, USA) solution 100uL in 3.9mL DMEM/F12, and the concentration of the collagenase type II is 312.5U/mL;
the tissue is digested by collagenase to achieve the purpose of separating cells, a large number of primary cells are obtained after digestion in a short time, and the activity of the cells is not affected.
(3.2) adding trypsin (gibco, USA) into the obtained precipitate for secondary digestion, incubating and digesting for 30min at 37 ℃, adding an equal volume of sterile cell protection solution to stop digestion, filtering with a 100-mesh filter screen, collecting filtrate and centrifuging, wherein the centrifugal force is 3000 Xg, and centrifuging for 8-10 min; discarding the supernatant, and keeping the cell precipitate;
the volume ratio of trypsin to precipitate is preferably 1: 1, 4. the concentration of trypsin is preferably 0.25%;
the formula of the cell protection solution is as follows: 20% FBS (gibco, USA), 1% double antibody (Hyclone, USA), DMEM/F12(gibco, USA) to make up the balance by volume; for example, 50mL of the cell protective solution was prepared by adding 10mL of LFBS and 0.5mL of the diabody to 39.5mL of the MEM/F12.
(3.3) resuspending the cell pellet obtained in the step (3.2) by using a sterile cell protection solution, uniformly inoculating the cell pellet into a T-75 culture flask, and changing the solution after 2-3 days and after 5-6 days;
(4) cell passage and seed preservation and freezing: when the cell fusion reaches more than 80%, digesting and passaging, uniformly dividing the cell suspension into sterile culture bottles, supplementing cell protective solution, and adding 5% (volume ratio) CO at 37 deg.C2And (4) continuously culturing in the environment, wherein after inoculating for 2-3 days, the cell fusion can reach 80% -90%, and performing passage and cryopreservation to complete the separation culture of the mesenchymal stem cells.
Preferably, in the method for extracting mesenchymal stem cells from umbilical cord Wharton's jelly tissue, the passage cryopreservation in the step (4) is: resuspending the cells in a freezing medium, freezing in a programmed cooling box (Biosharp, China) at-80 deg.C for 24 hr, and storing in liquid nitrogen for a long period; the frozen stock solution is prepared by mixing 90 percent of FBS and 10 percent of DMSO (Yuan, X., et al. Mesenchym cell thermal indexes FLT3L and CD1c (+) polymeric cells in system cellulose sheets natures. Nat Commun 2019.10(1):2498.) in volume ratio, namely 1mL of frozen stock solution is prepared by 0.9mL of FBS (gibco, USA) and 0.1mL of LDMSO (Sigma, USA).
After culturing the P0 Mesenchymal stem Cells isolated by the method, detecting the surface markers of the flow Cells by using CD34-FITC, CD44-APC, CD45-APC, CD90-PE, CD73-PE and CD105-FITC labeled antibodies (the detection method is disclosed in Beravolu, N., et al. isolation and Characterization of Mengchial Cells from Human Umbilical corp and Fetal Place. J. Vis Exp 2017.122; Araujo, A.B., et al. isolation of Human sensory stem Cells from cell, chorion, planta and clinical code of biological Cord: composition of folk biological protocols 359945 (35998) and CD 3526 of low expression); high-level expression of CD44, CD90, CD73 and CD105 proves that the obtained mesenchymal stem cells have good trilineage differentiation potential, namely osteogenesis, adipogenesis and chondrogenesis.
The beneficial effect of this application is as follows:
(1) the method disclosed by the invention can be used for removing the umbilical cord adventitia and the expected blood vessel, so that a pure Wharton's jelly tissue can be separated, pollution in the separation process can be effectively avoided, the purity of the finally obtained umbilical cord mesenchymal stem cells can be ensured, and the obtained mesenchymal stem cells are large in quantity, high in purity and strong in proliferation and differentiation capacity.
(2) The invention utilizes collagenase solution containing high concentration type II collagenase (the conventional use concentration is 250U/mL), and effectively improves the separation efficiency on the premise of ensuring the cell proliferation and differentiation activity. After the enzyme concentration is properly increased, the cell separation efficiency is greatly improved, a larger amount of primary cells can be obtained, and the proliferation and differentiation activity of the cells is high.
(3) The invention uses two proteases for digestion, shortens enzyme action time while ensuring effective digestion of umbilical cord tissue blocks, reduces the influence of chemical components on the activity state of tissue cells as much as possible, obtains cells with large quantity, high purity and strong multiplication capacity, and greatly improves the separation rate of mesenchymal stem cells.
(4) According to the invention, high-concentration FBS is used for culturing primary cells (the formula of the conventional cell protection solution is DMEM/F12+ 10% FBS + 1% double antibody), so that the primary cells can obtain high nutrition after being isolated, the cell activity is maintained, and the cell proliferation and differentiation are ensured.
Drawings
FIG. 1 is a detailed flow chart of the culture protocol of example 1.
FIG. 2 is a photograph showing adherent culture of tissue pieces of Wharton's jelly of comparative example 1.
Fig. 3 is a cell fusion curve of the mesenchymal stem cells of the generation of P0 in example 1 and comparative example 1 in different time periods.
FIG. 4 is the proliferation curve of the mesenchymal stem cells of the generations P0-P5 of example 1.
FIG. 5 is a photograph showing the P0 generation cells isolated by the method described in example 1 and comparative example 1, cultured for 2 days.
FIG. 6 is a photograph showing the P0 generation cells isolated by the method described in example 1 and comparative example 1, cultured for 5 days.
Fig. 7 is a graph showing the flow cytometry detection result of the mesenchymal stem cells of generation P0 obtained in example 1.
Fig. 8 is a graph of the results of osteogenic induction staining and alizarin red control staining detection.
FIG. 9 is a graph of the results of the detection of adipogenic induced staining versus the control staining of oil red "O".
FIG. 10 is a graph showing the results of the detection of chondrogenic induction staining and Aliskine blue control staining.
Detailed Description
The present invention will be further described below in order to better explain the concept and advantages of the present invention. Unless otherwise indicated, the experimental procedures in the following examples are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer (product description).
Examples reagents, media sources/formulations:
trypsin was purchased from gibco, usa;
cell protection solution: the cell protection solution comprises 20% FBS (gibco, USA) and 1% double antibody (Hyclone, USA) by volume ratio, and the balance is made up by DMEM/F12(gibco, USA); for example, 50mL of the cell protective solution was prepared by adding 10mL of LFBS and 0.5mL of the diabody to 39.5mL of the MEM/F12.
Collagenase solution: the collagenase is prepared by dissolving 12500U/mL type II collagenase (Sigma, USA) solution 100uL in 3.9mLDMEM/F12, wherein the concentration of the type II collagenase is 312.5U/mL;
is prepared by dissolving type II collagenase in DMEM/F12, wherein the type II collagenase concentration is 312.5U/mL
Cell protection solution: DMEM/F12+ 20% FBS + 1% double antibody
Freezing and storing liquid: the frozen stock solution is prepared by mixing 90% FBS and 10% DMSO in volume ratio, namely 1mL of frozen stock solution is prepared by 0.9mL FBS (gibco, USA) and 0.1mL LDMSO (Sigma, USA); the preparation method of the frozen stock solution can also be seen in the publications "Yuan, X., et al, Mesenchyl cell thermal indexes FLT3L and CD1c (+) polymeric cells in system lipids erythromatoses tissues Nat Commun 2019.10(1): 2498.";
procedural cooling box: purchased from Biosharp, china;
FBS-containing complete medium: DMEM/F12+ 20% FBS + 1% double antibody by volume.
Example 1:
as shown in figure 1, a method for extracting mesenchymal stem cells from umbilical cord Wharton's jelly tissue comprises the following steps:
(1) collecting, storing and transporting umbilical cords: obtaining an umbilical cord under an aseptic condition, preliminarily cleaning human umbilical cord internal arterial venous blood, ligating two ends of the umbilical cord by using rubber bands, soaking the umbilical cord in aseptic normal saline, storing and transporting at a low temperature (0-4 ℃), and processing within 6 hours;
(2) separating the tissue of the Wharton's jelly: repeatedly washing with sterile normal saline to remove umbilical cordSurface and blood vessel residual blood, then using sterile scissors to divide the umbilical cord into segments of about 3-4cm, then placing the segments of umbilical cord on sterile gauze soaked by normal saline, using ophthalmic scissors to longitudinally cut off umbilical cord adventitia tissues from the superficial layer, fixing one side of adventitia, using an operating knife to scrape and strip Wharton's jelly and adventitia, then using hemostatic forceps to remove blood vessel, and ensuring tissue moistening in the process; finally, washing the separated Wharton's jelly tissue by using sterile normal saline, and shearing the Wharton's jelly tissue into pieces of 1-3mm3Tissue mass;
(3) cell extraction and culture:
(3.1) adding a collagenase solution into the tissue blocks cut in the step (2) for carrying out primary digestion, wherein the volume ratio of the added collagenase solution to the tissue blocks is 1: 1;
incubating and digesting at 37 deg.C for 30min, centrifuging at 3000 Xg for 8min, discarding supernatant, and collecting precipitate;
(3.2) adding trypsin (with the concentration of 0.25%) into the precipitate obtained in the step (3.1) for secondary digestion, incubating and digesting for 30min at 37 ℃, adding an equal volume of sterile cell protection solution to stop digestion, filtering by a 100-mesh filter screen, collecting filtrate, centrifuging, wherein the centrifugal force is 3000 Xg, and centrifuging for 8 min; discarding the supernatant, and keeping the cell precipitate;
the volume ratio of trypsin to precipitate was 1: 1;
(3.3) resuspending the cell sediment obtained in the step (3.2) by using a sterile cell protection solution, uniformly inoculating the cell sediment into a T-75 culture flask, wherein cells adherent overnight are P0 generation cells, replacing the cell protection solution after 2 days, and completely replacing the cell protection solution after 5 days;
(4) cell passage and seed preservation and freezing: when the cell fusion reaches more than 80%, digesting and passaging, uniformly dividing the cell suspension into sterile culture bottles, supplementing cell protective solution, and adding 5% (volume ratio) CO at 37 deg.C2And (4) continuously culturing in the environment, wherein after inoculating for 2-3 days, the cell fusion can reach 80% -90%, and performing passage and cryopreservation to complete the separation culture of the mesenchymal stem cells.
In this embodiment, the passage cryopreservation means: after being resuspended in the freezing medium, the cells are frozen and stored in a programmed cooling box at minus 80 ℃ for 24 hours and then transferred into liquid nitrogen for long-term storage.
Comparative example 1: extraction and culture of mesenchymal stem cells by climbing method
A method for extracting mesenchymal stem cells from umbilical cord Wharton's jelly tissue comprises the following steps:
(1) collecting, storing and transporting umbilical cords: obtaining an umbilical cord under an aseptic condition, preliminarily cleaning the internal arterial venous blood of the umbilical cord, ligating two ends of the umbilical cord by using rubber bands, soaking the umbilical cord in aseptic normal saline, storing and transferring the umbilical cord at a low temperature, and treating the umbilical cord within 6 hours;
(2) repeatedly washing with sterile normal saline, removing residual blood on the surface of the umbilical cord and in blood vessels, dividing the umbilical cord into segments of about 3-4cm by using sterile scissors, placing the segments of the umbilical cord on sterile gauze soaked by normal saline, longitudinally cutting off umbilical cord adventitial tissues from a shallow layer by using an ophthalmic scissors, fixing one side of the adventitia, scraping and stripping Wharton's jelly and the adventitia by using a scalpel, and then removing the blood vessels by using hemostatic forceps, wherein tissue moistening is ensured in the process; finally, washing the separated Wharton's jelly tissue by using sterile normal saline, and shearing the Wharton's jelly tissue into pieces of 1-3mm3A size tissue mass;
(3) cell culture: placing 15-20 tissue blocks into T-75 culture flask, adding appropriate amount of FBS-containing complete culture medium, and culturing at 37 deg.C under 5% CO2Culturing in the environment, changing liquid after 3-4 days, changing liquid completely after 6 days, and changing liquid every 3-4 days, wherein the picture of adherent culture is shown in figure 2;
(4) cell passage and seed preservation and freezing: when the cell fusion reaches more than 80%, digesting and passaging (the cells before passaging are defined as P0 generation cells), uniformly distributing the cell suspension into a sterile culture bottle, and supplementing fresh FBS-containing complete culture medium at 37 ℃ and 5% CO2The culture is continued in the environment. The harvested large amount of unused umbilical cord mesenchymal stem cells can be resuspended by using a freezing medium, frozen and stored in a programmed cooling box at-80 ℃ for 24 hours and then transferred into liquid nitrogen for long-term storage.
Example 2 cell culture assay
The growth conditions of the P0 generation mesenchymal stem cells obtained in example 1 and comparative example 1 were observed and cell fusion degree curves were drawn at different time periods, and the results are shown in fig. 3, which shows that the cell fusion is faster in the method of example 1.
Cell counting was performed according to the mesenchymal stem cells of the P0-P5 generation of example 1 and a growth curve was plotted, and the results are shown in fig. 4.
The cells cultured for 3 days in the P0 generation and the cells cultured for 6 days in the P0 generation in example 1 and comparative example 1 were selected and photographed under a microscope, as shown in FIG. 5, A is example 1 and B is comparative example 1, and it can be seen that about 20% of the cells adhered to example 1 by the culture to day 2, but no cell climbing-out was seen in comparative example 1.
Images of example 1 and comparative example 1 after 5 days of P0 generation culture were observed under a microscope, as shown in fig. 6, a is example 1, and B is comparative example 1; it can be seen that the degree of cell fusion of example 1 has reached more than 80%, whereas the cells of comparative example 1 are only about 1% adherent.
After culturing the mesenchymal stem cells of generation P0 obtained in example 1, analyzing the expression of the cell surface marker by flow assay, the results are shown in fig. 7, and in fig. 7, a-F are CD90, CD45, CD105, CD73, CD44 and CD34 respectively; therefore, the human umbilical cord mesenchymal stem cells obtained by the method in example 1 have high purity, and positive expression of CD73, CD105, CD90 and CD44 is realized; CD45 and CD34 are expressed negatively (see the literature: Beravolu, N., et al, isolation and Characterization of sensory chemical Cells from Human Ulmbrial Cord and Total plant. J. Vis Exp 2017.122; Araujo, A.B., et al, isolation of Human sensory stem Cells from biomanion, chorion, central differentiation and systemic code: composite of viral fermentation protocols. Biotechnol Lett 2018.40(6):989 + 998.), which indicates that the differentiated stem Cells obtained in example 1 have a better capacity.
The mesenchymal stem cells obtained in example 1 were cultured, and then the cells of P3 generation were subjected to three-line differentiation and identification, and then the cells were subjected to osteogenesis (Cyagen, Cat.: HUXUC-90021), adipogenesis (Cyagen, Cat.: HUXUC-90031) and chondrogenesis (Cyagen, Cat.: HUXUC-90042) induced differentiation and staining according to the specific procedures of the human umbilical cord mesenchymal stem cell induced differentiation medium kit (Cyagen, USA), and the results are shown in FIGS. 8-10: fig. 8 shows osteogenic induction staining (fig. 8B) and alizarin red control staining (fig. 8A), fig. 9 shows adipogenic induction staining (fig. 9B) and oil red "O" control staining (fig. 9A), and fig. 10 shows chondrogenic induction staining (fig. 10B) and alistic blue control staining (fig. 10A) (Dominici, m., et al. minimal criterion for defining porous structural cells. the International Society for Cellular Therapy condition, Cytotherapy 2006.8(4): 315:317), it can be seen that the human umbilical cord mesenchymal stem cells obtained by the method of example 1 have good multidirectional differentiation potential, and can be directionally differentiated into osteocytes, chondrocytes, adipocytes.
The experimental results prove that the two-step enzyme digestion method applied in the embodiment 1 has simple process, greatly shortens the preparation period of primary cells, obtains the cells with good activity, strong proliferation capacity, good multidirectional differentiation potential and high separation efficiency, and provides sufficient cell sources for large-scale experimental research and clinical application.
The technical route and the advantages of the invention are shown above, and the invention is more specific and detailed. It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of this invention and is not to be considered as limiting thereof, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (8)

1. A method for separating and culturing human umbilical cord mesenchymal stem cells is characterized by comprising the following specific steps:
1) obtaining an umbilical cord under an aseptic condition, cleaning the internal arterial venous blood of the umbilical cord, ligating two ends of the umbilical cord by using rubber bands, and soaking the umbilical cord in aseptic normal saline;
2) repeatedly washing umbilical cord with sterile normal saline to remove residual blood on umbilical cord surface and blood vessel, dividing umbilical cord into 3-4cm segments, scraping and peeling Wharton's jelly and adventitia, washing separated Wharton's jelly tissue with sterile normal saline, and dividing into 1-3mm segments3The tissue mass of (a);
3.1) adding collagenase solution into the tissue block, incubating for 30min at 37 ℃, centrifuging and taking the precipitate;
the collagenase solution is prepared by dissolving 12500U/mL type II collagenase solution 100uL in 3.9mL DMEM/F12;
3.2) adding trypsin into the precipitate, incubating for 30min at 37 ℃, adding a cell protection solution to stop digestion, filtering, collecting filtrate, centrifuging, and taking cell precipitate;
the formula of the cell protection solution is as follows: 20% of FBS, 1% of double antibody and DMEM/F12 for making up the balance in terms of volume ratio;
3.3) resuspending the cell precipitate with a cell protection solution and uniformly inoculating the cell precipitate into a culture flask, replacing the cell protection solution after 2 to 3 days, and completely replacing the cell protection solution after 5 to 6 days;
when the cell fusion reaches more than 80%, uniformly distributing the cell suspension into a sterile culture bottle, supplementing cell protective solution, and keeping the temperature at 37 ℃ under 5% CO2And (4) continuously culturing in the environment, and when the cell fusion reaches 80-90%, carrying out passage freezing storage, thus completing the separation culture of the mesenchymal stem cells.
2. The isolated culture method of human umbilical cord mesenchymal stem cells according to claim 1, wherein in the step 3.1), the volume ratio of the collagenase solution to the tissue mass is 1: 1.
3. the isolated culture method of human umbilical cord mesenchymal stem cells according to claim 1, wherein the centrifugation in step 3.1) is 3000 Xg for 8-10 min.
4. The isolated culture method of human umbilical cord mesenchymal stem cells according to claim 1, wherein the centrifugation in step 3.2) is 3000 Xg for 8-10 min.
5. The isolated culture method of human umbilical cord mesenchymal stem cells according to claim 1, wherein the concentration of trypsin in step 3.2) is 0.25%.
6. The isolated culture method of human umbilical cord mesenchymal stem cells according to claim 5, wherein the volume ratio of trypsin to precipitate in step 3.2) is 1: 1.
7. the isolated culture method of human umbilical cord mesenchymal stem cells according to any one of claims 1 to 6, wherein the step 4) of subculturing is to resuspend the cells in a freezing medium, cryopreserve the cells in a programmed cooling box at-80 ℃, and store the cells in liquid nitrogen for a long time after 24 hours.
8. The isolated culture method of human umbilical cord mesenchymal stem cells according to claim 7, wherein the frozen stock solution is obtained by mixing 90% FBS and 10% DMSO in volume ratio.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111690601A (en) * 2020-06-08 2020-09-22 海南优尼科尔生物科技有限公司 Preparation method of umbilical cord mesenchymal stem cell preparation
CN114214276A (en) * 2021-12-31 2022-03-22 武汉光谷中源药业有限公司 Off-the-shelf human umbilical cord-derived mesenchymal stem cells and preparation method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111690601A (en) * 2020-06-08 2020-09-22 海南优尼科尔生物科技有限公司 Preparation method of umbilical cord mesenchymal stem cell preparation
CN114214276A (en) * 2021-12-31 2022-03-22 武汉光谷中源药业有限公司 Off-the-shelf human umbilical cord-derived mesenchymal stem cells and preparation method and application thereof

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