CN108060116B - Extraction, separation and culture method of fetal rat endothelial progenitor cells - Google Patents
Extraction, separation and culture method of fetal rat endothelial progenitor cells Download PDFInfo
- Publication number
- CN108060116B CN108060116B CN201810147120.2A CN201810147120A CN108060116B CN 108060116 B CN108060116 B CN 108060116B CN 201810147120 A CN201810147120 A CN 201810147120A CN 108060116 B CN108060116 B CN 108060116B
- Authority
- CN
- China
- Prior art keywords
- cells
- pancreatin
- endothelial progenitor
- culture
- fetal rat
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/069—Vascular Endothelial cells
- C12N5/0692—Stem cells; Progenitor cells; Precursor cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Microbiology (AREA)
- Vascular Medicine (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a separation culture method of fetal rat lung source endothelial progenitor cells, which comprises the steps of putting a pregnant uterus of a pregnant rat into precooled PBS with double resistance of penicillin and streptomycin for soaking, taking out the pregnant rat, putting the pregnant rat into a dish of precooled PBS with double resistance of penicillin and streptomycin, and washing; removing lung tissue, adding PBS, crushing, digesting the crushed tissue with 0.25% pancreatin, culturing in DMEM medium containing 10% FBS, centrifuging, collecting precipitate, suspending in EBM-2 medium containing growth factor and 2% FBS serum, and culturing in 10% FBS serum6the/ML cell concentration is inoculated in a 6-well plate, the plate is placed in a constant temperature incubator for culture, when the growth and fusion of primary cells reach 80 to 90 percent, the primary cells are digested and subcultured by 0.25 percent of pancreatin until the cells present a uniform shape, and the culture is finished.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a method for extracting, separating and culturing endothelial progenitor cells, in particular to a method for extracting, separating and culturing endothelial progenitor cells from fetal rats.
Background
ENDOTHELIAL PROGENITOR CELLS (ENDOTHELIAL PROGENITOR CELLS, abbreviated as "EPCS") are vascular ENDOTHELIAL precursor CELLS that can be directionally differentiated into mature ENDOTHELIAL CELLS, also known as angioblasts. When the organism is ischemic, EPCS can mobilize from bone marrow to enter peripheral blood, and reaches ischemic tissues to participate in angiogenesis. The EPCS cultured and amplified in vitro is delivered into the body, so as to improve the quantity and quality of the EPCS in circulation and promote the angiogenesis of ischemic parts. Therefore, EPCS transplantation is a new approach for the treatment of ischemic vascular diseases.
For the extraction of endothelial progenitor cells, the literature reports that umbilical cord blood or bone marrow and peripheral blood of animals of different ages after birth are mainly collected, mononuclear cells are obtained by a density gradient centrifugation method, and then the mononuclear cells are inoculated into a culture bottle or a culture dish coated by special substances for in vitro induction culture.
The key to the clinical application of the endothelial progenitor cells is to search for an ideal tissue source, but the methods for the recruitment, isolation and culture of the endothelial progenitor cells have large difference, and the obtained endothelial progenitor cells have small quantity, low amplification efficiency and slow cell proliferation. The invention adopts a new source way to extract the endothelial progenitor cells, namely adopts the lung tissues of the fetal rat as the tissue source, and provides a new feasible method for extracting, separating and culturing the endothelial progenitor cells.
Disclosure of Invention
The invention aims to provide a method for extracting, separating and culturing fetal rat lung-derived endothelial progenitor cells.
The invention relates to a method for extracting, separating and culturing fetal rat lung-derived endothelial progenitor cells, which comprises the following steps:
(1) taking lungs of a fetal rat, putting the lungs into precooled PBS containing 500U-1000U of penicillin and streptomycin double antibody, and crushing lung tissues;
(2) digesting the disrupted tissue of step (1) with pancreatin;
(3) after digestion, culturing the cells in DMEM medium containing FBS for 15-18H;
(4) then taking out non-adherent cells, centrifuging and taking out the sediment;
(5) suspending the precipitate of step (4) with EBM-2 medium containing serum of growth factor and FBS, inoculating in a pore plate at a concentration, and culturing in an incubator;
(6) changing the solution every 2-4 days, when the growth and fusion of primary cells reach 80-90%, digesting and subculturing by using pancreatin, and continuously subculturing until the cells present a uniform shape to finish the culture.
The method of the present invention described above, the fetal rat, the method of obtaining the fetal rat, comprises: (1) anesthetizing pregnant mouse, taking out the beaded pregnant uterus, and soaking in precooled PBS containing 500-1000U penicillin and streptomycin dual-antibody; (2) separating umbilical cord tissue connecting the navel and placenta of the fetal rat from the soaked pregnant uterus, putting the fetal rat into a dish containing 500U-1000U of precooled PBS with double resistance of high-strength penicillin and streptomycin, and washing to obtain the fetal rat.
In a specific embodiment, the method for extracting, separating and culturing fetal mouse lung-derived endothelial progenitor cells of the invention is realized by the following embodiments, comprising the following steps:
(1) anesthetizing a pregnant mouse, taking out a beaded pregnant uterus, and soaking in precooled PBS containing 500-1000U of penicillin and streptomycin double antibody;
(2) separating umbilical cord tissues connecting the navel and the placenta of the fetal rat from the soaked pregnant uterus, putting the fetal rat into a dish containing precooled PBS (phosphate buffer solution) with each 500U-1000U of penicillin and streptomycin double resistance, and washing;
(3) taking out the lung of a fetal rat, putting the lung into precooled PBS containing 500U-1000U of penicillin and streptomycin double antibody, and crushing lung tissues;
(4) digesting the disrupted tissue of step (3) with 0.25% pancreatin;
(5) after digestion, 16H was cultured in DMEM medium containing 10% FBS;
(6) then taking out non-adherent cells, centrifuging and taking out the sediment;
(7) suspending the pellet of step (6) in EBM-2 medium containing growth factor and serum of 2% FBS at 106The cells were seeded in 6-well plates at 37 ℃ in 5% CO2Culturing in a constant-temperature incubator;
(8) changing the solution every 3 days, digesting and subculturing with 0.25% of pancreatin when the primary cells grow and fuse to 80-90%, and continuously subculturing until the cells show uniform morphology, thereby completing the culture.
In the above embodiment, in the step (1), the soaking is performed for 3-5 minutes, and the pregnant mouse is a pregnant mouse with a gestational age of 15-19 days; washing in the step (2), wherein the washing frequency is 2 times; the size of the broken tissue in the step (3) is 0.5-1.0MM3(ii) a The pancreatin digestion in the step (4) adopts a stepwise digestion method, and each step is placed in a cell culture box at 37 ℃ for incubation for 10MIN until the tissues are completely digested; the centrifugation in step (6) was 1000RPM,5 MIN.
In a specific embodiment, a method for extracting, separating and culturing fetal rat lung-derived endothelial progenitor cells comprises the following steps:
(1) the pregnant mouse with the gestational age of 15-19 days is anesthetized by 10% chloral hydrate, immediately placed into 75% sterilized alcohol for soaking for 15 minutes after anesthesia, moved into cells, the abdomen of the pregnant mouse is upward, and the limbs are fixed.
(2) Shearing the abdominal epidermis and the mucous layer of the pregnant mouse layer by using sterile ophthalmic scissors and tweezers, taking out the beaded pregnant uterus, soaking the beaded pregnant uterus in precooled PBS containing 500U-1000U of high-strength penicillin and streptomycin double-antibody, and then moving the pregnant mouse into a clean bench.
(3) Cutting uterus with ophthalmological scissors, removing amniotic fluid, removing amniotic membrane, separating umbilical cord tissue connecting navel of fetal mouse and placenta, placing fetal mouse in a dish containing 500U-1000U precooled PBS with high-strength penicillin and streptomycin dual-resistance, and washing for 2 times.
(4) The lungs of fetal mice were carefully removed with ophthalmic scissors and forceps and placed in a dish containing 500U-1000U of a precooled PBS of a double antibody against penicillin and streptomycin, and cut into pieces one by one.
(5) Digesting the tissue of step (4) with 0.25% pancreatin by stepwise digestion, each of which was incubated in a 37 ℃ cell incubator for 10MIN until the tissue was completely digested.
(6) After digestion, 16H was cultured in DMEM medium containing 10% FBS.
(7) Then, non-adherent cells were removed at 1000RPM for 5MIN, centrifuged, and the pellet was removed.
(8) Suspending the pellet of step (7) in EBM-2 medium containing growth factor and serum (2% FBS) at 10%6The cells were seeded in 6-well plates at 37 ℃ in 5% CO2Culturing in a constant temperature incubator.
(9) Changing the solution every 3 days later, digesting and subculturing with 0.25% of pancreatin when the primary cells grow and fuse to 80-90%, and continuously subculturing until the cells show a uniform shape, thereby completing the culture.
The endothelial progenitor cells cultured by the method are identified by the following method:
1) the morphological observation of the endothelial progenitor cells,
2) identifying EPCS surface markers by cellular immunofluorescence, such as CD34, CD133, CD31 and VEGFR-2 antigen,
3) flow cytometry identification of EPCS surface markers: CD34, CD133, CD31, VEGFR-2 antigen,
4) the laser confocal detection of the phagocytic function of the EPCS: the ability to take DIL-ACLLDL and bind FITC-UEA-1,
5) EPCS in vitro lumen formation experiment: and (3) matrigel.
The endothelial progenitor cells cultured by the method of the invention are subjected to growth and proliferation capacity detection: such as CCK 8.
In the method of the invention, the gestational age of the pregnant mouse in the step (1) is preferably 15-19 days, the gestational age is too small, the fetal mouse is not mature, the lung is small, and the separation is difficult; uterus, fetal mouse and lung tissues separated from the pregnant body in the steps (2), (3) and (4) are preferably put into PBS containing high-strength dual-resistance antibiotics for soaking and washing: the applicable concentration of the double-resistant penicillin and the streptomycin is 500U-1000U, and the soaking time is 5-10 minutes; in the steps (6) and (8), the double antibody with the concentration of 500U-1000U not only can effectively inhibit bacteria, but also has no side effect on cells;
in the method of the invention, in the step (4), the lung tissue is cut into 0.5-1.0MM3, the smaller the tissue block is, the more complete the pancreatin digestion is, and the higher the cell climbing efficiency after inoculation is; and (6) uniformly paving the digested tissue suspension in a pore plate, wherein a small amount of culture medium is suitable. The culture medium has a large amount, and fine tissue blocks are suspended and are not easy to adhere to the wall, so that the yield of primary cells is greatly reduced;
the method of the invention establishes a method for extracting, separating and culturing fetal rat lung-derived endothelial progenitor cells. The method has the following 6 advantages:
(1) the method is simple and easy to implement and has strong operability;
(2) the cell amplification efficiency is high, the cell can be seen to climb out after the tissue is attached to the wall for 24-48H, and the primary digestion can be carried out after the tissue grows for 8-12 days generally. The cells are uniformly expressed when continuously transmitted to P5-P7 generations due to different fetal rat individuals, and then the cells obtained by the separation culture method are confirmed to be endothelial progenitor cells through immunophenotyping, growth characteristics, multidirectional differentiation and the like;
(3) the proliferation capacity of the cells is strong, the cells still have the activity of endothelial progenitor cells after 5 generations, and the proliferation and differentiation capacity is maintained, so that the purified cells P2-P5 are preferably used, and the number of the cells available in the experiment is sufficient;
(4) compared with the endothelial progenitor cell extraction and separation culture technology widely applied at present, the endothelial progenitor cell derived from fetal rat lung has the advantages that: the separation method is easy to implement, the amplification efficiency is high, the multiplication capacity is abnormally strong, the passage time is long, and the cell quantity is large; particularly, in the aspect of CCK8 for detecting cell proliferation, the proliferation activity of fetal rat-derived endothelial progenitor cells is obviously higher than that of endothelial progenitor cells derived from bone marrow, peripheral blood and umbilical cord blood.
(5) Provides an ideal new cell source for the transplantation of the endothelial progenitor cells.
In conclusion, the method for extracting and separating and culturing the fetal rat lung-derived endothelial progenitor cells has great application prospect and value.
Drawings
FIG. 1 morphology of isolated cultured endothelial progenitor cells;
FIG. 2 shows that the endothelial progenitor cells cultured separately strongly express CD34, CD133, CD31 and VEGFR-2;
FIG. 3 shows the result of phagocytic function test of isolated cultured endothelial progenitor cells;
FIG. 4 shows the results of in vitro tube formation experiment for detecting and identifying the tube forming ability of EPCS to form a tube cavity-like structure;
FIG. 5 example 2 shows the morphology of lung, heart and liver derived endothelial progenitor cells 1D isolated and cultured.
Detailed Description
The following examples used the following materials and sources, respectively:
cleaning grade SD rats (male and female) were from the experimental animals center of chongqing university of medicine, 0.25% trypsin (AMRESCO corporation), phosphate buffered saline PBS, DMEM medium from HYCLONE, fetal bovine serum FBS from GIBCO, cell culture dish (diameter 10CM), cell culture 6-well plate from CORNING; CD34, CD133, CD31, VEGF-R2, IGG-FITC, and IGG-PE control flow antibodies were purchased from BD, alkaline phosphatase (ALP) staining kit from Shanghai Sun biosciences, and oil Red O from SIGMA.
The present invention is further illustrated by the following specific examples. The examples were carried out under conventional conditions, without specifying the conditions.
Example 1 extraction, isolation and culture of fetal murine Lung-derived endothelial progenitor cells
The implementation steps are as follows:
(1) healthy adult clean grade SD rats (female/male) were purchased at the experimental animal center of the university of chongqing medicine, 2 female rats and 1 male rats were in the same rat box overnight at the time of estrus of the rats, the next morning the vaginal opening of the female rat was checked, the presence of a sperm plug was visually recognized to indicate conception, and the gestational age was calculated (day 0). If the check is negative, the caging is continued until the check is positive.
(2) The pregnant mouse with the gestational age of 15-19 days is anesthetized by 10% chloral hydrate, immediately placed into 75% sterilized alcohol for soaking for 15 minutes after anesthesia, moved into cells, the abdomen of the pregnant mouse is upward, and the limbs are fixed.
(3) Shearing the abdominal epidermis and mucosa layer by layer of pregnant mouse with sterile ophthalmic scissors and forceps to fully expose pregnant uterus, taking out the beaded uterus with the ophthalmic scissors and forceps, soaking in precooled PBS containing 500U-1000U high-strength penicillin and streptomycin, and transferring into a super clean bench.
(4) Cutting uterus with sterile ophthalmic scissors, removing amniotic fluid, removing amniotic membrane layer, separating umbilical cord tissue connecting navel and placenta of fetal rat, separating each fetal rat (containing placenta, with number of 10-16), placing fetal rat into a dish containing 500U-1000U precooled PBS with high-strength penicillin and streptomycin, and washing for 2 times.
(5) The lungs of fetal mice were carefully removed with ophthalmic scissors and forceps and placed in a dish containing 500U-1000U of pre-cooled PBS of the dual-antibody penicillin and streptomycin, and individually minced to a fine tissue mass of 0.5-1.0MM 3.
(6) Digesting the tissue in step (4) with 0.25% pancreatin by stepwise digestion, each step being incubated in a 37 ℃ cell incubator for 10MIN until the tissue is completely digested.
(7) After termination of digestion, 16H was cultured in DMEM medium containing 10% FBS.
(8) After 16H, non-adherent cells were removed at 1000RPM for 5MIN, centrifuged, and the pellet was removed.
(9) The pellet in step (8) was suspended with EBM-2 medium containing growth factor and serum (2% FBS) at 106The cells were seeded in 6-well plates at 37 ℃ in 5% CO2Culturing in a constant temperature incubator.
(10) Changing the culture solution every 3 days later, digesting and subculturing with 0.25% of pancreatin when the primary cells grow and fuse to 80-90%, and continuously subculturing until the cells show uniform morphology, thereby completing the culture. And the following authentication or verification is made.
Verification example 1
The morphological observation of the endothelial progenitor cells obtained by separation and culture of the invention:
(1) fetal mouse lung-derived endothelial progenitor cells isolated using the method of example 1: climbing out primary (P0 generation) cells after tissue adherence for 24-48H, continuously culturing for 3-4 days, observing the cells with an inverted microscope to have heterogeneous shapes, long fusiform shapes, polygonal shapes and round shapes (see figure 1);
(2) following the passaging procedure described in example 1, and subject to individual differences in fetal mice, cells were observed under an inverted microscope as they passed through P2-P2 passages and appeared to be relatively uniform long fusiform. The cell morphology did not change significantly when the cell was continuously passed over 5 generations (see FIG. 1).
(3) The morphological results of the fetal rat lung-derived endothelial progenitor cells show that: the cells separated and cultured by the invention have the characteristic of long fusiform shape of endothelial progenitor cells and the characteristics of adherent growth and repeated passage.
Verification example 2
The immunophenotyping of the endothelial progenitor cells isolated and cultured by the invention:
(1) selecting fetal mouse lung-derived endothelial progenitor cells obtained by separation and passage in example 1 (such as P2 generation), counting after 0.25% pancreatin digestion, subpackaging into 6 tubes at 1 × 105/tube, washing with PBS for 1 time, and resuspending the cell pellet with 50 μm L PBS;
(2) to each tube was added 1 μm L of the corresponding flow antibody: CD34, CD133, CD31, VEGFR-2, FITC-IGG, PE-IGG, incubated at 4 ℃ for 30 minutes in the absence of light;
(3) PBS was washed 1 time, detected on flow cytometer and analyzed.
(4) The immunophenotyping analysis result of the fetal rat lung-derived endothelial progenitor cells shows that: the cells separated and cultured by the invention strongly express CD34, CD133, CD31 and VEGFR-2 and meet the surface marker characteristics of endothelial progenitor cells (see figure 2).
Verification example 3
The phagocytic function of the endothelial progenitor cells obtained by separation and culture of the invention is analyzed:
(1) fetal mouse lung-derived endothelial progenitor cells obtained by isolation and passage as described in example 1 (e.g., passage P2) were counted after 0.25% of trypsinization, and seeded on a slide glass for cell slide.
(2) DIL-ACLLDL and FITC-UEA-1 were incubated for 12H.
(3) And (5) observing the fluorescence condition under a laser confocal microscope.
(4) The result of phagocytic function analysis of fetal rat cardiovascular endothelial progenitor cells shows that: when cells were observed under a confocal laser microscope by double-fluorescent staining with DIL-ACLLDL and FITC-UEA-1, the cells were considered to be EPCS, which was capable of exciting both red and green fluorescence and was synthesized as yellow fluorescence. Red fluorescence was AC-LDL positive cells, green ueA-1 positive cells and double positive cells EPCS (FIG. 3).
Verification example 4
The invention analyzes the lumen forming ability of endothelial progenitor cells obtained by separation and culture:
(1) fetal mouse lung-derived endothelial progenitor cells obtained by isolation and passage (e.g., passage P2) were selected according to the method of example 1 and counted after 0.25% of trypsinization.
(2) Cells were seeded on MATRIGL matrigel plates.
(3) The result of the analysis of the tube cavity forming capability of the fetal rat lung-derived endothelial progenitor cells shows that: it can be seen that the cells cultured for 14 days can form a lumen-like structure, which is consistent with the endothelial characteristics of endothelial progenitor cells (see fig. 4).
Verification example 5
The proliferation capacity of the endothelial progenitor cells obtained by isolated culture is analyzed as follows:
(1) selecting fetal mouse lung-derived endothelial progenitor cells obtained by separation and passage in the method of example 1 (such as P2 generation), carrying out 0.25% pancreatin digestion, then re-suspending the cell pellet with a quantitative culture medium, counting the cells, and adjusting the concentration to 1 × 104/ML;
(2) inoculating cell suspension into 96-well plate (100 μm L per well), and culturing in cell culture box at constant temperature of 37 deg.C and saturated humidity of 5% CO 2;
(3) taking 3-well cells every day, digesting each well with quantitative 0.25% pancreatin, counting the cells, and replacing the undigested cells with liquid 1 time every 3 days;
(4) continuously counting for 3 days, and drawing a growth curve graph;
the above examples are merely illustrative of some preferred embodiments of the present invention, which are described in more detail and detailed, but the scope of the present invention is not limited to the above embodiments. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Example 2 comparison of extraction, isolation, culture and comparison of fetal murine Lung, Heart and hepatic derived endothelial progenitor cells
The extraction, separation and culture method comprises the following steps:
(1) taking SD pregnant mouse, soaking with 75% ethanol immediately after anesthesia, transferring into cells, and fixing rat limbs on foam board on the table surface with pins.
(2) The abdomen was dissected open, uterus and fetal rats were carefully removed, transferred to a clean bench, placenta, amniotic fluid, amnion, etc. were removed from fetal rats, and the fetal rats were placed in a dish with double-antibody pre-cooled PBS.
(3) PBS wash 2 times. The heart, lung, liver and other organs of the fetal rat were carefully removed.
(4) The organs are classified and cut into pieces, each tissue piece is digested by 0.25% pancreatin, and the stepwise digestion method is adopted until the tissues are complete.
(5) After termination of digestion, heart, lung tissue, liver-derived cells were cultured in DMEM medium containing 10% FBS for 16H.
(6) Non-adherent cells were removed individually, 1000RPM,5MIN, and centrifuged.
(7) EBM-2 medium containing growth factor and serum (2% FBS) was inoculated into 6-well plates at 106/ML cell concentration, and cultured in a 5% CO2 incubator at 37 ℃ to obtain endothelial progenitor cells derived from heart, lung tissue and liver, respectively.
Morphological observation of Endothelial Progenitor Cells (EPCS) derived from heart, lung tissue and liver obtained by culture was carried out by the method of test example 1. The results found the worst, hardly adherent liver. From the morphology, the lung-derived endothelial progenitor cells are better, the heart adheres more, but some hybrid cells exist, and the morphology of the heart-derived cells and EPCs are not much like and are too long, which is shown in FIG. 5, and shows that the lung-derived endothelial progenitor cells are optimal.
Claims (9)
1. A separation culture method of fetal rat lung-derived endothelial progenitor cells comprises the following steps:
(1) taking lungs of a fetal rat, putting the lungs into precooled PBS containing 500U-1000U penicillin and streptomycin double antibody, and crushing lung tissues;
(2) digesting the disrupted tissue of step (1) with pancreatin;
(3) after digestion, culturing for 15-18 hours by using DMEM medium containing FBS;
(4) then taking out non-adherent cells, centrifuging and taking out the sediment;
(5) suspending the precipitate in the step (4) by using an EBM-2 culture medium containing serum of a growth factor and FBS, inoculating the suspension in a pore plate, and culturing in an incubator;
(6) changing the solution every 2-4 days, digesting and subculturing the primary cells by pancreatin when the growth and fusion of the primary cells reach 80-90%, and continuously subculturing until the cells are in a uniform shape to finish the culture;
wherein the obtaining of the fetal rat comprises:
(A) anesthetizing pregnant mouse, taking out the beaded pregnant uterus, and soaking in precooled PBS containing 500-1000U penicillin and streptomycin dual-antibody;
(B) separating umbilical cord tissue connecting the navel and placenta of the fetal rat from the soaked pregnant uterus, putting the fetal rat into a dish containing 500U-1000U of precooled PBS with double resistance of high-strength penicillin and streptomycin, and washing to obtain the fetal rat.
2. The method according to claim 1, wherein the washing in step (2) is performed 2 times.
3. The method of claim 1, wherein the pancreatin in step (2) is 0.25% pancreatin, and the DMEM medium in step (3) contains 10% FBS for 16 hours.
4. The method of claim 1, wherein the centrifugation in step (4) is 1000rpm for 5min and the EBM-2 medium in step (5) contains 2% FBS.
5. The method of claim 1, wherein the seeding of step (5) is carried out at a cell concentration of 106A constant temperature chamber containing 5% CO2The temperature is 37 ℃, the solution is changed every 3 days in the step (6), and the concentration of the pancreatin is 0.25 percent.
6. The method of claim 1, wherein the pancreatin digestion in step (2) is performed by stepwise digestion, and each step is incubated in a 37 ℃ cell culture chamber for 10min until the tissue is completely digested.
7. The method of claim 1, wherein said soaking in step (a) is for a time period of 3 to 5 minutes.
8. The method according to claim 1, wherein the pregnant mouse in step (a) is a pregnant mouse with gestational age of 15-19 days.
9. The method of claim 1, wherein the size of the disrupted tissue is 0.5-1.0mm in step (1)3。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810147120.2A CN108060116B (en) | 2018-02-12 | 2018-02-12 | Extraction, separation and culture method of fetal rat endothelial progenitor cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810147120.2A CN108060116B (en) | 2018-02-12 | 2018-02-12 | Extraction, separation and culture method of fetal rat endothelial progenitor cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108060116A CN108060116A (en) | 2018-05-22 |
| CN108060116B true CN108060116B (en) | 2020-03-10 |
Family
ID=62134452
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810147120.2A Expired - Fee Related CN108060116B (en) | 2018-02-12 | 2018-02-12 | Extraction, separation and culture method of fetal rat endothelial progenitor cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108060116B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101265464A (en) * | 2008-05-13 | 2008-09-17 | 中国人民解放军第二军医大学 | Method for separating and cultivating porcine marrow endothelial progenitor cell |
| WO2011073521A1 (en) * | 2009-12-15 | 2011-06-23 | Petri Salven | Methods for enriching adult-derived endothelial progenitor cells and uses thereof |
| CN106754650A (en) * | 2017-02-24 | 2017-05-31 | 哈尔滨中科赛恩斯生物技术有限公司 | A kind of endothelial progenitor cells cultural method of derived from bone marrow |
-
2018
- 2018-02-12 CN CN201810147120.2A patent/CN108060116B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101265464A (en) * | 2008-05-13 | 2008-09-17 | 中国人民解放军第二军医大学 | Method for separating and cultivating porcine marrow endothelial progenitor cell |
| WO2011073521A1 (en) * | 2009-12-15 | 2011-06-23 | Petri Salven | Methods for enriching adult-derived endothelial progenitor cells and uses thereof |
| CN106754650A (en) * | 2017-02-24 | 2017-05-31 | 哈尔滨中科赛恩斯生物技术有限公司 | A kind of endothelial progenitor cells cultural method of derived from bone marrow |
Non-Patent Citations (4)
| Title |
|---|
| 1种新来源的内皮祖细胞提取分离方法;李丽玲等;《免疫学杂志》;20190331;第35卷(第3期);第269-276页 * |
| 一种新来源的内皮祖细胞提取分离方法及SIRT6对B细胞发育及BCR信号的影响;李丽玲;《中国优秀硕士学位论文全文数据库》;20190531;第1-65页 * |
| 内皮克隆形成细胞的研究进展;吴晓欣等;《中南大学学报》;20151231;第40卷(第5期);第564-567页 * |
| 小鼠内皮祖细胞的培养和移植;杨悦等;《中国组织工程研究与临床康复》;20091008;第31卷(第41期);第8109-8113页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108060116A (en) | 2018-05-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN108685948B (en) | Preparation method of medical cell repairing agent | |
| JP6711757B2 (en) | Method to differentiate into hepatocytes using pluripotent stem cells derived from mesenchymal stem cells | |
| US9211306B2 (en) | Cellular therapeutic agent for incontinence or urine comprising stem cells originated from decidua or adipose | |
| CN112430567B (en) | Culture method and application of urine-derived renal stem cells | |
| CN109893541B (en) | Application of exosome derived from menstrual blood stem cells in preparation of medicine for treating intrauterine adhesion | |
| You et al. | The biological characteristics of human third trimester amniotic fluid stem cells | |
| RU2433172C2 (en) | Method of obtaining homogenous population of stem cells and its application | |
| CN107974429A (en) | A kind of method and Optimal Medium of quick separating culture human airway epithelial cells | |
| JP7370613B2 (en) | Methods for producing hair follicles and de novo papillae and their use for in vitro testing and in vivo transplantation | |
| CN108220229A (en) | A kind of preparation method for improving umbilical cord derived mesenchymal stem cell primary cell yield | |
| CN108373990B (en) | Culture medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes and method thereof | |
| CN104762257A (en) | Method for preparing mesenchymal stem cell from umbilical cord | |
| CN110090227B (en) | Use of human amniotic epithelial cells in treatment of graft-versus-host disease | |
| CN114874981A (en) | Mesenchymal stem cell exosome and application thereof in immune cell culture | |
| KR20120006386A (en) | Stem cell derived from first trimester placenta and cellular therapeutic agents comprising the same | |
| JP6711756B2 (en) | Method for differentiating into adipocytes using pluripotent stem cells derived from mesenchymal stem cells | |
| CN106701669A (en) | Mesenchymal stem cell for clinical treatment as well as preparation method and application thereof | |
| CN105112359A (en) | Separating and cultivating method of mouse umbilical cord mesenchymal stem cells | |
| CN108060116B (en) | Extraction, separation and culture method of fetal rat endothelial progenitor cells | |
| CN110731970A (en) | cell preparation for treating allergic rhinitis | |
| CN102119936B (en) | Method for preparing injection for treating ischemic brain damage by using human amniotic mesenchymal cells and injection | |
| CN110484491B (en) | Method for obtaining amniotic membrane and amniotic fluid derived endothelial progenitor cells and purification culture method thereof | |
| CN109771697B (en) | Dermal fibroblast skin sheet and construction method and application thereof | |
| CN114591890B (en) | In-vitro model for simulating interaction of resistance blood vessel smooth muscle cells and endothelial cells and construction method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200310 Termination date: 20220212 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |