CN111575229B - Separation method of placenta decidua stem cells - Google Patents
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Abstract
The invention relates to a separation method of placental decidua stem cells, belonging to the technical field of regenerative medicine and biology. The separation method of the decidua placenta stem cells comprises the following steps: collecting placenta, and soaking in PBS (basal medium) containing 1X-2X double antibody and 10% high-glucose DMEM; stripping the bottom molting film and cleaning; cutting the decidua basalis, carrying out enzymolysis by using a DMEM high-glycosyl basal medium containing 0.25% of pancreatin and 0.02% of EDTA in the enzymolysis solution a, centrifuging, removing the supernatant, and carrying out enzymolysis by using a DMEM high-glycosyl basal medium containing 0.1-0.2mg/mL of collagenase IV, 0.01-0.05mg/mL of neutral enzyme, 5-10ng/mL of Fas ligand inhibitor and 5-10ng/mL of survivin inhibitor in the enzymolysis solution b; then subpackaging, counting and inoculating. The separation method of the placental decidua stem cells adopts a double enzymolysis method to extract the placental decidua stem cells, and has the advantages of simple operation steps, high cell acquisition rate, sufficient extracted cell number and high cell viability rate. Furthermore, the present invention aims to extract the decidua basalis stem cells of the placenta in all directions.
Description
Technical Field
The invention relates to a separation method of placental decidua stem cells, belonging to the technical field of regenerative medicine and biology.
Background
With the ongoing and intensive research on Mesenchymal Stem Cells (MSCs), MSC transplantation has become an effective means of treating many systemic diseases. In recent years, the placenta has become an important source of MSCs of interest to numerous scholars, and scholars have succeeded in isolating MSCs with transplantation potential from the amniotic membrane and chorion of the fetal side of the placenta, but studies on the decidua basalis of the maternal side are rare. The decidua basalis stem cells extracted from the placenta have the advantages of rich content, low rejection reaction after transplantation, no ethical barrier and the like. The decidua basalis mesenchymal stem cells have strong proliferation capacity, the multiplication time is faster than that of mesenchymal stem cells from bone marrow, fat and chorion reported in other documents, and the cell cycle is similar to that of mesenchymal stem cells from other sources.
At present, the separation and extraction method of placental decidua stem cells mainly adopts an enzyme digestion method, and although the enzyme digestion method can obtain sufficient cells, the content of the placental decidua stem cells is low. In addition, enzymolysis digestion extraction is carried out on the decidua part at the bottom of the placenta, the size of a digested tissue is large, the time consumption is long, the number of obtained cells is large, but the survival rate of stem cells is low; extracting stem cells from decidua basalis, wherein the obtained cells contain a large amount of hybrid cells, which causes troubles for subsequent purification; the whole extraction process of decidua stem cells at the bottom has complicated steps, the fluctuation of the number of the extracted cells is large, the pollution probability is greatly increased, and the extraction process is not beneficial to industrial production.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a separation method of placental decidua basalis stem cells, the method adopts a novel enzymolysis scheme, the cell activity is obviously improved, the extracted cells are sufficient, and the number of the decidua basalis stem cells contained in the cells is high.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for separating placental decidua stem cells comprises the following steps:
(1) collecting placenta, and soaking in placenta preservation solution;
(2) carrying out basal decidua stripping on the placenta in the step (1) to obtain a basal decidua, preliminarily cleaning the basal decidua by using cleaning solution, and then sucking the cleaning solution by using a disposable pipette to clean again until basal decidua blood is cleaned;
(3) shearing the decidua basalis treated in the step (2) by using scissors, performing enzymolysis on the ground decidua basalis by using an enzymolysis liquid a, centrifuging, removing supernate, and performing enzymolysis by using an enzymolysis liquid b;
(4) taking out the tissue fluid subjected to enzymolysis in the step (3), subpackaging the tissue fluid into a centrifuge tube, adding a cell cleaning solution, balancing and centrifuging;
(5) pouring out the supernatant centrifuged in the step (4), adding a cell cleaning solution to resuspend cell precipitates, filtering the cell precipitates into a centrifuge tube by using a cell screen, uniformly mixing, taking cell suspension, counting by using a cell counting plate, balancing and centrifuging;
(6) and (4) pouring out the supernatant obtained after centrifugation in the step (5), adjusting the cell inoculation density by using a culture medium solution, adding the double antibody, and uniformly mixing and inoculating.
The inventors of the present invention found through research that: the key point of the enzymolysis method for extracting the placental decidua stem cells is to improve the extraction quantity of the stem cells and ensure the survival rate of the stem cells. Therefore, in the method for separating placental stem cells according to the present invention, the composition of the enzymatic hydrolysate and the extraction conditions become important factors. Before the invention, the placenta amnion is the main extraction tissue of the placenta stem cell; the amnion is mechanically sheared and then is subjected to enzymolysis extraction by enzyme liquid, the obtained cells are sufficient, but the number of the contained hybrid cells is large, and the number of stem cells is low. The invention aims to optimize the components of the enzymolysis liquid, increase the nutrition supply and improve the anti-apoptosis property of cells. In addition, the cell pollution rate extracted by the separation method of the placental stem cells is extremely low; the separation method provided by the invention is simple and effective in the whole operation engineering, and the separation and extraction efficiency is obviously improved; meanwhile, the conditions of extraction and separation are optimized, and the survival rate of the decidua basalis stem cells is improved as much as possible.
As a preferred embodiment of the method for isolating placental stem cells according to the present invention, the step (1) is specifically performed by immersing the placenta obtained after parturition in a preservation solution at a temperature of 4 ℃ within 6 hours; the placenta preservation solution is a basal culture medium containing 1 x-2 x double antibody and high-glucose DMEM with the volume concentration of 10% in PBS.
The invention selects the placenta of healthy delivery, adopts low-temperature placenta preserving fluid to preserve the placenta, enters a test process after 6 hours, and extracts hematopoietic stem cells with high activity as far as possible in a short time.
In a preferred embodiment of the method for isolating placental stem cells according to the present invention, in step (2), the washing solution for preliminarily washing decidua basalis is 0-4 ℃ PBS, and the volume of the disposable pipette is 10 mL.
As a preferred embodiment of the method for isolating placental stem cells according to the present invention, in step (3), the area of the ground decidua is 3X 3cm after being minced2(ii) a Performing enzymolysis with 200R at 37 deg.C at volume ratio of 10:1 for 0.5h, centrifuging at 1500rpm for 5 min; performing enzymolysis on the enzymolysis liquid b and the decidua basalis at a volume ratio of 20:1 for 1.5-2.5h at the temperature of 37 ℃ under 200R.
As a preferred embodiment of the method for separating placental stem cells according to the present invention, the enzymatic hydrolysate a is a DMEM high-sugar basic medium containing pancreatin at a mass concentration of 0.25% and EDTA at a mass concentration of 0.02%; the enzymolysis liquid b is a DMEM high-glucose basal medium containing 0.1-0.2mg/mL collagenase IV, 0.01-0.05mg/mL neutral enzyme, 5-10ng/mL Fas ligand inhibitor and 5-10ng/mL survivin inhibitor.
The placenta preservation solution contains nutrient substances such as umbilical plasma and the like, so that the nutrient supply of placenta tissues is ensured; survivin inhibitor (anti-apoptosis reagent) is added, so that cell starting apoptosis program is greatly inhibited, the cell survival rate is increased, DMEM high-sugar basic culture medium in enzymolysis liquid is used as solvent, nutrition supply is provided for cells in the enzymolysis process, and the cell vitality is ensured.
In a preferred embodiment of the method for separating placental stem cells according to the present invention, in step (4), the tissue fluid is removed and dispensed into 4 50mL centrifuge tubes, wherein the volume of the tissue fluid in each centrifuge tube is not more than 25mL, the volume of the cell wash solution added is the same as the volume of the tissue fluid, the centrifugation rate is 2000rpm, and the centrifugation time is 5 min.
As a preferred embodiment of the method for separating placental stem cells, in step (5), a pipette is used to add 10mL of cell washing solution into each tube to resuspend cell pellets, a 100 μm cell screen is used to filter the cell pellets into 150 mL centrifuge tubes, after mixing, a pipette tip is used to take 50-200 μ L of cell suspension, and a cell counting plate is used to count the cell suspension; the speed of the centrifugation was 2000rpm and the time of the centrifugation was 5 min.
As a preferred embodiment of the method for separating the placental stem cells, the counting on the cell counting plate is specifically that after the cell suspension and 0.4% trypan blue are uniformly mixed in a ratio of 1:1, 10 mu L of the mixture is dripped on the cell counting plate to be counted, the cell activity is detected, and the total number of the cells is calculated. Wherein the 0.4% trypan blue is purchased commodity with a brand of sigma, a batch number of RNBG4319 and a commodity number of T8154-100 mL.
As a preferred embodiment of the method for isolating placental stem cells according to the present invention, in step (6), the medium is Lonza complete medium, andthe cell inoculation density is (1-1.5) multiplied by 105cell/mL, and the double antibody is 100 multiplied by the double antibody.
As a preferred embodiment of the method for isolating placental stem cells according to the present invention, in step (6), the step of inoculating with a 10cm culture dish comprises inoculating 10mL of cell suspension with a pipette into the 10cm culture dish; the step of inoculating with a 15cm culture dish is to inoculate 20mL of cell suspension with a pipette into the 15cm culture dish; after inoculation, the culture dish is shaken for 5 times in a cross way, so that the cell suspension is uniformly distributed in the culture dish.
The separation method of the placental stem cells adopts a double enzymolysis method to extract the decidua cells at the bottom of the placenta, and has the advantages of simple operation steps, high cell acquisition rate, sufficient extracted cell number and high cell survival rate. Furthermore, the present invention aims to extract the decidua basalis cells of the placenta in all directions.
Compared with the prior art, the invention has the beneficial effects that:
(1) the separation method of the placental stem cells has the advantages of simplified operation steps, short experimental operation time and low pollution probability of the extracted cells;
(2) the separation method of the placental stem cells has the advantages that the number of the extracted cells is sufficient, the content of the stem cells is greatly improved, and the cell survival rate is high;
(3) the number of cells extracted by the separation method of the placental stem cells is stable.
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FIG. 1 is a microscopic observation image of cell morphology of cells extracted by the separation method of decidua placenta stem cell of the invention example 1;
FIG. 2 is a microscopic observation image of cell morphology of cells extracted by the separation method of decidua placenta stem cell of the invention example 2;
FIG. 3 is a microscopic observation image of cell morphology of cells extracted by the separation method of decidua placenta stem cell of the invention example 3;
FIG. 4 is a view showing the observation of cell morphology under a microscope of cells extracted by the method for separating placental decidua stem cells according to comparative example 1;
FIG. 5 is a microscopic observation image of cell morphology of cells extracted by the method for separating placental decidua stem cells described in comparative example 2.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
The 0.4% Trypan blue described in the following examples of the present invention is a purchased commodity under the brand name of sigma, the lot number of RNBG4319, and the commodity number of T8154-100 mL.
Example 1
This embodiment is a method for separating placental stem cells according to the present invention, comprising the following steps:
(1) collecting placenta, and soaking in placenta preservation solution: soaking placenta obtained after delivery in PBS preservation solution of basal medium containing 1X-2X double antibody and high-glucose DMEM with volume concentration of 10% within 6 hr, and preserving and transporting at 4 deg.C;
(2) carrying out basal decidua stripping on the placenta in the step (1) to obtain a basal decidua, preliminarily cleaning the basal decidua of the placenta by using PBS (phosphate buffer solution) at 0 ℃, and then sucking cleaning solution into tissues by using a 10mL disposable pipette until basal decidua blood is cleaned;
(3) cutting the decidua basalis processed in the step (2) into pieces with the area of 3 multiplied by 3cm by using scissors2Carrying out enzymolysis on the ground decidua by using enzymolysis liquid a (a DMEM high-sugar base culture medium containing pancreatin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%) and the decidua at the volume ratio of 10:1 for 0.5h at the temperature of 37 ℃, and centrifuging at 1500rpm for 5 min; performing enzymolysis for 1.5h at 37 ℃ by 200R with enzymolysis liquid b (a DMEM high-sugar base medium containing 0.1mg/mL collagenase IV, 0.01mg/mL neutral enzyme, 5ng/mL Fas ligand inhibitor and 5ng/mL survivin inhibitor) and a basal decidua in a volume ratio of 20: 1;
(4) taking out the tissue fluid subjected to enzymolysis in the step (3), subpackaging the tissue fluid into 4 centrifuge tubes with the volume of 50mL (each tube is not more than 25mL), adding equal volume of cell cleaning fluid, balancing, and centrifuging for 5min at 2000rpm in a centrifuge;
(5) slightly pouring off the supernatant centrifuged in the step (4), adding 10mL of cell cleaning solution into each tube by using a pipette to resuspend cell sediment, filtering the cell sediment into 150 mL centrifuge tubes by using a 100-micron cell screen, uniformly mixing, taking 50 mu L of cell suspension by using a pipette tip, counting the cell suspension by using a cell counting plate, balancing the centrifuge tubes, and centrifuging the centrifuge tubes for 5min at 2000 rpm; the cell counting method comprises the following steps: according to the cell suspension: mixing 0.4% trypan blue (volume ratio) 1:1, dripping 10 μ L onto a cell counting plate, counting, detecting cell activity and calculating total number of cells;
(6) the supernatant after centrifugation in step (5) was decanted, and the cell seeding density was adjusted to 1X 10 with Lonza complete medium solution based on the cell count results5cell/mL, adding 100 Xdouble antibody according to volume, mixing uniformly and inoculating; wherein, the step of inoculating with a 10cm culture dish comprises inoculating 10mL of cell suspension into the 10cm culture dish by using a pipette; the step of inoculating with a 15cm culture dish is to inoculate 20mL of cell suspension with a pipette into the 15cm culture dish; after inoculation, the culture dish is shaken for 5 times in a cross way, so that the cell suspension is uniformly distributed in the culture dish.
Example 2
This embodiment is a method for separating placental stem cells according to the present invention, comprising the following steps:
(1) collecting placenta, and soaking in placenta preservation solution: soaking placenta obtained after delivery in PBS preservation solution of basal medium containing 1X-2X double antibody and high-glucose DMEM with volume concentration of 10% within 6 hr, and preserving and transporting at 4 deg.C;
(2) carrying out basal decidua stripping on the placenta in the step (1) to obtain a basal decidua, preliminarily cleaning the basal decidua of the placenta by using PBS (phosphate buffer solution) at 4 ℃, and then sucking cleaning solution into tissues by using a 10mL disposable pipette until basal decidua blood is cleaned;
(3) cutting the decidua basalis processed in the step (2) into pieces with the area of 3 multiplied by 3cm by using scissors2Carrying out enzymolysis on the ground decidua by using enzymolysis liquid a (a DMEM high-sugar base culture medium containing pancreatin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%) and the decidua at the volume ratio of 10:1 for 0.5h at the temperature of 37 ℃, and centrifuging at 1500rpm for 5 min; enzymatic hydrolysate b (containing 0.2mg/mL collagenase IV, 0.05mg/mL neutral enzyme, 10ng/mL Fas ligand inhibitor and 10 ng/based on the total weight of the cells) was used at 37 ℃DMEM high-glucose basal medium of ml survivin inhibitor) and the decidua basalis in a volume ratio of 20:1, performing 200R enzymolysis for 2.5 h;
(4) taking out the tissue fluid subjected to enzymolysis in the step (3), subpackaging the tissue fluid into 4 centrifuge tubes with the volume of 50mL (each tube is not more than 25mL), adding equal volume of cell cleaning fluid, balancing, and centrifuging for 5min at 2000rpm in a centrifuge;
(5) slightly pouring off the supernatant centrifuged in the step (4), adding 10mL of cell cleaning solution into each tube by using a pipette to resuspend cell sediment, filtering the cell sediment into 150 mL centrifuge tubes by using a 100-micron cell screen, uniformly mixing, taking 200 mu L of cell suspension by using a pipette tip, counting the cell suspension by using a cell counting plate, balancing the centrifuge tubes, and centrifuging the centrifuge tubes for 5min at 2000 rpm; the cell counting method comprises the following steps: according to the cell suspension: mixing 0.4% trypan blue (volume ratio) 1:1, dripping 10 μ L onto a cell counting plate, counting, detecting cell activity and calculating total number of cells;
(6) the supernatant after centrifugation in step (5) was discarded, and the cell seeding density was adjusted to 1.5X 10 using a Lonza complete medium solution based on the cell count results5cell/mL, adding 100 Xdouble antibody according to volume, mixing uniformly and inoculating; wherein, the step of inoculating with a 10cm culture dish comprises inoculating 10mL of cell suspension into the 10cm culture dish by using a pipette; the step of inoculating with a 15cm culture dish is to inoculate 20mL of cell suspension with a pipette into the 15cm culture dish; after inoculation, the culture dish is shaken for 5 times in a cross way, so that the cell suspension is uniformly distributed in the culture dish.
Example 3
This embodiment is a method for separating placental stem cells according to the present invention, comprising the following steps:
(1) collecting placenta, and soaking in placenta preservation solution: soaking placenta obtained after delivery in PBS preservation solution of basal medium containing 1X-2X double antibody and high glucose DMEM with quality name of 10% within 6 hr, and storing and transporting at 4 deg.C;
(2) carrying out basal decidua stripping on the placenta in the step (1) to obtain a basal decidua, preliminarily cleaning the basal decidua of the placenta by using PBS (phosphate buffer solution) at the temperature of 2 ℃, and then sucking cleaning solution into tissues by using a 10mL disposable pipette until basal decidua blood is cleaned;
(3) by scissorsCutting the decidua basalis processed in the step (2) into pieces with the area of 3 multiplied by 3cm2Carrying out enzymolysis on the ground decidua by using enzymolysis liquid a (a DMEM high-sugar base culture medium containing pancreatin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%) and the decidua at the volume ratio of 10:1 for 0.5h at the temperature of 37 ℃, and centrifuging at 1500rpm for 5 min; performing enzymolysis for 2h at 37 ℃ by 200R with enzymolysis liquid b (a DMEM high-sugar base medium containing 0.4mg/mL collagenase IV, 0.1mg/mL neutral enzyme, 15ng/mL Fas ligand inhibitor and 15ng/mL survivin inhibitor) and a bottom decidua membrane in a volume ratio of 20: 1;
(4) taking out the tissue fluid subjected to enzymolysis in the step (3), subpackaging the tissue fluid into 4 centrifuge tubes with the volume of 50mL (each tube is not more than 25mL), adding equal volume of cell cleaning fluid, balancing, and centrifuging for 5min at 2000rpm in a centrifuge;
(5) slightly pouring off the supernatant centrifuged in the step (4), adding 10mL of cell cleaning solution into each tube by using a pipette to resuspend cell sediment, filtering the cell sediment into 150 mL centrifuge tubes by using a 100-micron cell screen, uniformly mixing, taking 100-micron L of cell suspension by using a pipette tip, counting the cell suspension by using a cell counting plate, balancing the centrifuge tubes, and centrifuging the centrifuge tubes for 5min at 2000 rpm; the cell counting method comprises the following steps: according to the cell suspension: mixing 0.4% trypan blue (volume ratio) 1:1, dripping 10 μ L onto a cell counting plate, counting, detecting cell activity and calculating total number of cells;
(6) the supernatant after centrifugation in step (5) was discarded, and the cell seeding density was adjusted to 1.5X 10 using a Lonza complete medium solution based on the cell count results5cell/mL, adding 100 Xdouble antibody according to volume, mixing uniformly and inoculating; wherein, the step of inoculating with a 10cm culture dish comprises inoculating 10mL of cell suspension into the 10cm culture dish by using a pipette; the step of inoculating with a 15cm culture dish is to inoculate 20mL of cell suspension with a pipette into the 15cm culture dish; after inoculation, the culture dish is shaken for 5 times in a cross way, so that the cell suspension is uniformly distributed in the culture dish.
Comparative example 1
This comparative example was carried out with reference to the method for isolating and culturing decidua placenta and mesenchymal stem cells of umbilical cord described in document 1, as follows:
washing placenta tissue with sterile saline, and carefully separating umbilical cord and placentaAnd (3) a basal decidua tissue, and separating the umbilical cord from the basal decidua tissue. Shearing sterile tissue into small tissue blocks, digesting with collagenase and pancreatin, filtering the digested mixed liquid with 200 mesh sieve, inoculating into cell culture bottle, culturing with DF12 culture solution containing 10% fetal calf serum at 37 deg.C and 5% CO2Culturing in an incubator, digesting the cells by pancreatin when the cells grow to 80% fusion, and carrying out passage according to the ratio of 1: 3.
The document 1 is: korean wave, wang tao, wang billow, chi glu, yanzhoxin, and yue ru, mengyi, yangming, korean chio the isolation of mesenchymal stem cells from human placental decidua and their biological characteristics study [ J ] journal of chinese experimental hematology, 2013, (3).
Comparative example 2
The comparative example is carried out by referring to the isolated culture method of human placental decidua mesenchymal stem cells described in document 2, and specifically includes the following steps:
after signing informed consent with a parturient and family members, placenta of a healthy human full-moon parturient (from obstetrics and gynecology department of the Zhujiang hospital, southern medical university) is taken, a bottom periostracum layer on the side of a placenta maternal body is cut off under aseptic conditions to be rice grain-like, after being sequentially digested by 0.1% type II collagenase and 0.25% trypsin, a human lymphocyte separation solution (with the density of 1.077g/L) is centrifuged at 2000r/min for 20min, a middle leucoderma layer is sucked, after being washed twice by PBS, the placenta is resuspended by a DMEM/F12 culture solution containing 10% FBS, and the placenta is inoculated into a 25T culture bottle according to the density of 1 × 106/L. Culturing in a saturated humidity incubator with 5% CO2 at 37 ℃, changing the culture solution for the first time after 7 days, and then changing the culture solution for 1 time every 3-4 days. Digesting with 0.25% trypsin when cell fusion reaches about 70%, passaging at a ratio of 1: 2 or 1:3, and observing cell morphology change under a periodically inverted microscope
The document 2 is: luzhonghui, zhangshi loyal, chengqiang, wangxuefeng, lufenuron fly, liujian, prun, and li heroic.
Experimental example 1
Cell number extraction and viability assay
Division of placental decidua Stem cells according to examples 1 to 3 of the present inventionThe separation method is used for extracting the number and the activity of the cells, and the detection method comprises the following steps: taking freshly separated placental decidua stem cells, adjusting cell density to 1 × 106cell/mL. According to the cell suspension: after 0.4% trypan blue 3:1(v: v) was mixed well, 20. mu.L of the mixed cell solution was added to a cell counting plate, and the cell viability and volume were measured by a Countstar cell counter.
The number and activity of cells extracted by the method for culturing mesenchymal stem cells of placental decidua as described in comparative example 1 and comparative example 2 were determined according to the methods described in the corresponding literatures.
The results of the quantity extraction and viability test of the cells P0 in examples 1-3 and comparative examples 1-2 of the present invention are shown in Table 1 below, and the cell morphology observation under microscope is shown in FIGS. 1-5.
TABLE 1 detection of the number and viability of cells extracted by the separation method of placental decidua stem cells according to the present invention
| Examples | Number of viable cells | Total number of cells | Rate of cell viability |
| Example 1 | 4.69×108cells | 4.77×108cells | 98.2％ |
| Example 2 | 5.21×108cells | 5.33×108cells | 97.6％ |
| Example 3 | 5.57×108cells | 5.83×108cells | 95.4％ |
| Comparative example 1 | 2.96×108cells | 3.48×108cells | 85.1％ |
| Comparative example 2 | 2.73×108cells | 3.16×108cells | 86.3％ |
As can be seen from the above table, the present invention extracts the decidua basalis stem cells from placenta at 4.69X 108～5.57×108Among cells, the cell activity is up to more than 95%; the cell extraction quantity and the cell viability rate are both higher than those of the comparative example 1 and the comparative example 2, which shows that the cell viability rate and the cell viability rate extracted by the double enzymolysis method are high.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A method for separating decidua stem cells at the bottom of placenta is characterized by comprising the following steps:
(1) collecting placenta, and soaking in placenta preservation solution;
(2) carrying out basal decidua stripping on the placenta in the step (1) to obtain a basal decidua, preliminarily cleaning the basal decidua by using cleaning solution, and then sucking the cleaning solution by using a disposable pipette to clean again until basal decidua blood is cleaned;
(3) shearing the decidua basalis treated in the step (2) by using scissors, performing enzymolysis on the ground decidua basalis by using an enzymolysis liquid a, centrifuging, removing supernate, and performing enzymolysis by using an enzymolysis liquid b; the area of the ground decidua after being cut into pieces is 3 multiplied by 3cm2(ii) a Performing enzymolysis with 200R at 37 deg.C at volume ratio of 10:1 for 0.5h, centrifuging at 1500rpm for 5 min; performing enzymolysis on the enzymolysis liquid b and the decidua basalis at the volume ratio of 20:1 for 1.5-2.5h at the temperature of 37 ℃ under 200R; the enzymolysis solution a is a DMEM high-sugar basic culture medium containing pancreatin with the mass concentration of 0.25% and EDTA with the mass concentration of 0.02%; the enzymolysis liquid b is a DMEM high-glucose basal medium containing 0.1-0.2mg/mL collagenase IV, 0.01-0.05mg/mL neutral enzyme, 5-10ng/mL Fas ligand inhibitor and 5-10ng/mL survivin inhibitor;
(4) taking out the tissue fluid subjected to enzymolysis in the step (3), subpackaging the tissue fluid into a centrifuge tube, adding a cell cleaning solution, balancing and centrifuging;
(5) pouring out the supernatant centrifuged in the step (4), adding a cell cleaning solution to resuspend cell precipitates, filtering the cell precipitates into a centrifuge tube by using a cell screen, uniformly mixing, taking cell suspension, counting by using a cell counting plate, balancing and centrifuging;
(6) and (4) pouring out the supernatant obtained after centrifugation in the step (5), adjusting the cell inoculation density by using a culture medium solution, adding the double antibody, and uniformly mixing and inoculating.
2. The separation method according to claim 1, wherein the step (1) is carried out by soaking the placenta obtained after delivery in a preservation solution at a temperature of 4 ℃ within 6 hours; the placenta preservation solution is a basal culture medium containing 1 x-2 x double antibody and high-glucose DMEM with the volume concentration of 10% in PBS.
3. The separation method of claim 1, wherein in step (2), the washing solution is 0-4 ℃ PBS, and the volume of the disposable pipette is 10 mL.
4. The separation method of claim 1, wherein in step (4), the volume of the interstitial fluid in each tube in the centrifuge tube is not more than 25mL, the volume of the added cell washing fluid is the same as the interstitial fluid, the speed of the centrifugation is 2000rpm, and the time of the centrifugation is 5 min.
5. The separation method of claim 1, wherein in step (5), 10mL of cell wash solution is added to each tube by a pipette to resuspend the cell pellet, the cell pellet is filtered by a 100 μm cell screen into 150 mL centrifuge tube, after mixing, 50-200 μ L of cell suspension is taken by a pipette tip and counted by a cell counting plate; the speed of the centrifugation was 2000rpm and the time of the centrifugation was 5 min.
6. The separation method according to claim 1 or 5, wherein the counting of the cell counting plate is carried out by mixing the cell suspension with 0.4% trypan blue at a ratio of 1:1, dripping 10 μ L of the mixture onto the cell counting plate, counting, detecting the viability of the cells and counting the total number of the cells.
7. The isolation method according to claim 1, wherein in the step (6), the medium is a Lonza complete medium, and the cell seeding density is (1-1.5). times.105cell/mL, and the double antibody is 100 multiplied by the double antibody.
8. The separation method according to claim 1, wherein in the step (6), the step of inoculating with a 10cm dish is inoculating 10mL of the cell suspension with a pipette into the 10cm dish; the step of inoculating with a 15cm culture dish is to inoculate 20mL of cell suspension with a pipette into the 15cm culture dish; after inoculation, the culture dish is shaken for 5 times in a cross way, so that the cell suspension is uniformly distributed in the culture dish.
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| KR20160121240A (en) * | 2015-04-10 | 2016-10-19 | 차의과학대학교 산학협력단 | Process for preparing immortalized mesenchymal stem cell line |
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