CN107663513B - Efficient separation and extraction method for uterine membrane stem cells and library building method - Google Patents
Efficient separation and extraction method for uterine membrane stem cells and library building method Download PDFInfo
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- CN107663513B CN107663513B CN201610600995.4A CN201610600995A CN107663513B CN 107663513 B CN107663513 B CN 107663513B CN 201610600995 A CN201610600995 A CN 201610600995A CN 107663513 B CN107663513 B CN 107663513B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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Abstract
The invention discloses a high-efficiency separation and extraction method and a reservoir building method for endometrial stem cells. The number of the uterine membrane stem cells obtained by separating the waste bottom sediment after density gradient centrifugation in the traditional method is about ten times that of the traditional leucoderma, and a uterine membrane stem cell bank can be established after the amplification culture of a large number of the harvested uterine membrane stem cells.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to separation and extraction of uterine membrane stem cells and establishment of a uterine membrane stem cell bank.
Background
In 1999, Science has rated the research result of human embryonic stem cells as the first of ten scientific and technological advances in the world in the present year, in 2000, Time has listed it as the first of ten scientific and technological achievements in the world after the end of the 20 th century, and it is considered that embryonic stem cells and human genome will simultaneously become the fields with the most development and application prospects in the new century. Stem cell research has become one of the most striking hotspots in the biological and medical fields in recent years. Stem Cells (SC) are a type of pluripotent cells with self-replicating ability (self-rejuvenating) that under certain conditions can differentiate into a variety of functional cells. At present, stem cell sources can be divided into embryonic stem cells and adult stem cells, but the two sources have partial limitations, so that the finding of the stem cell source for overcoming the defects of potential tumor formation, lack of specimen sources, ethical disputes and the like has important significance. In recent years, the U.S. Musina-led research group obtained isolated stem cells from menstrual blood of healthy women. The endometrial stem cells (MenSCs) avoid the defects of other stem cells and become a brand-new stem cell with great research and application potential.
However, according to the search of the prior art documents and patents (such as chinese patent application nos. 201110085328.4 and 201210116237.7), the density gradient centrifugation method is mainly used in the currently reported methods for obtaining the uterine membrane stem cells, but the tissue mass is filtered and removed, the traditional density gradient centrifugation method only obtains mononuclear cells of the leucoderma layer, the removed tissue mass is deposited and discarded at the bottom, and the number of the cells obtained by the method is small.
Disclosure of Invention
The invention aims to provide a high-efficiency separation and extraction method and a library construction method for uterine membrane stem cells, so as to overcome the defect of low yield of the separation and extraction method for the uterine membrane stem cells in the prior art.
According to the principle that the endometrium stem cells come from the exfoliated endometrium tissue, but not all the endometrium stem cells can become mononuclear cells and the exfoliated endometrium tissue block exists, the inventor continuously searches for the principle that the bottom sediment is washed and then adheres to the wall for culture, and the endometrium stem cells in the endometrium tissue block adhere to the wall in a clone shape, so that the number of the harvested stem cells is about 10 times of the number harvested by the traditional method, the efficiency of separating and extracting the endometrium stem cells is greatly improved, and a large amount of endometrium stem cell resources are provided for establishing a bank of the endometrium stem cells.
The technical scheme of the invention is as follows:
a high-efficiency separation and extraction method of uterine membrane stem cells comprises the steps of washing bottom sediment obtained after a menstrual blood sample is subjected to density gradient centrifugation, carrying out adherent culture, and harvesting the uterine membrane stem cells.
Preferably, the density gradient centrifugation is performed by slowly adding an equal volume of menstrual blood sample to the human lymphocyte separation solution, and centrifuging for 20-40 minutes at a temperature of 15-25 ℃ and a rotation speed of 300-500 g.
Preferably, the washing of the bottom precipitate is repeated three times by washing with 10 volumes of buffer.
Preferably, the menstrual blood sample refers to menstrual blood of the second day or the third day of the female menstrual cycle, and an equal volume of buffer containing one or more antibacterial substances is added. The buffer is also called menstrual blood preservative fluid.
Preferably, the antibacterial substance is one or more selected from amphotericin, penicillin, cefradine, cefazolin, cefadroxil, cefuroxime, cephalothin, streptomycin, tobramycin, tetracycline, oxytetracycline, erythromycin ethylsuccinate, clarithromycin, acetylspiramycin, azithromycin, roxithromycin, erythromycin, clindamycin and lincomycin.
Preferably, the adherent culture comprises a primary culture and a subculture expansion.
Preferably, the primary culture method of the utero-membrane stem cells is as follows: resuspending the target cells in the bottom sediment obtained after separation and washing with 10ml of alpha-MEM containing 10% -20% serum, placing the mixture in a plate, standing and culturing the mixture at 37 ℃ under the condition of 5% CO2, washing the mixture for 3 times with 10ml of buffer solution after 24 hours, replacing 10ml of fresh culture solution, and then replacing the solution once every 3 days; when the cells grew to about 80% -90%, they were digested and then passaged.
Preferably, the step of subculturing and expanding the uterine membrane stem cells comprises: and (3) completely replacing the fluid once every two days for the uteromembranous stem cells after passage in the primary culture method, digesting the uteromembranous stem cells after passage when the cells grow to be about 80-90%, and continuing passage.
The efficient uterine membrane stem cell banking method comprises the steps of menstrual blood collection, separation and extraction of uterine membrane stem cells, primary culture, subculture amplification, analysis and detection, long-term storage and periodic detection, wherein the steps of menstrual blood collection, separation, sterilization and extraction of uterine membrane stem cells, primary culture and amplification adopt any one of the methods.
Preferably, the assay comprises a sterility test, a flow cytometer test, a karyotype test, and a differentiation capacity test; the items for periodic detection comprise cell activity detection, sterility detection, flow cytometer detection and differentiation capacity detection after cell recovery.
Compared with the prior art, the invention has the following beneficial effects:
firstly, the waste bottom sediment of density gradient centrifugation is adopted to separate and extract the uterine membrane stem cells, the number of the harvested uterine membrane stem cells is about ten times that of the traditional leucoderma, and a uterine membrane stem cell bank can be established after the harvested large number of uterine membrane stem cells are amplified and cultured;
secondly, the invention adopts density gradient centrifugation, washing, adding antibacterial substances into the menstrual blood preserving fluid and other means to ensure the completeness of sterilization, so that the obtained uterine membrane stem cell product is not polluted.
Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.
Drawings
FIG. 1 is a photograph of density gradient centrifugation of uterine membrane stem cells according to an embodiment of the present invention;
FIGS. 2(a) and 2(b) are photographs of the adherent after the isolation and extraction of conventional uteromembranous stem cells as a comparative example (1 day);
FIGS. 3(a) and 3(b) are photographs of the anchorage-dependent cell wall after the isolation and extraction of the efficient endometrial stem cells according to the embodiment of the present invention (1 day);
FIGS. 4(a) and 4(b) are photographs of primary cells of a conventional uterine membrane stem cell isolation and extraction method as a comparative example (8 days);
FIGS. 5(a) and 5(b) are photographs of primary cells of the efficient isolating and extracting method for uteroconjunctival stem cells according to the embodiment of the invention (8 days);
FIGS. 6(a) and 6(b) are photographs (13 days) of P1 generation cells isolated and extracted from utero membrane stem cells as a conventional method of comparative example;
FIGS. 7(a) and 7(b) are photographs of P1 generation cells isolated and extracted from the uterine membrane stem cells of the efficient method according to the embodiment of the present invention (11 days).
Detailed Description
The invention provides a high-efficiency separation and extraction method of a uterine membrane stem cell, which selects bottom sediment (namely a uterine membrane tissue) after menstrual blood sample density gradient centrifugation, and performs adherent culture after washing to obtain a large amount of uterine membrane stem cells.
The principle of density gradient centrifugation is a method that the particles are respectively settled at a certain speed under the action of a certain centrifugal force by utilizing the difference of settlement coefficients of different particles, and zones are formed on different regions of the density gradient. The purpose of density gradient centrifugation is to remove a portion of bacteria, fungi and mycoplasma.
The density gradient centrifugation uses a cell separation solution (such as a human lymphocyte separation solution) with the density of 1.077, and the centrifugation is carried out at the rotating speed of 300-.
The menstrual blood sample refers to menstrual blood of the second day or the third day of the female menstrual cycle, and an equal volume of buffer containing various antibiotics is added, wherein the buffer is also called menstrual blood preservative fluid.
The menstrual blood preserving fluid can also be added with antibacterial substances; wherein the antibacterial substance is selected from one or more of amphotericin, penicillin, cefradine, cefazolin, cefadroxil, cefuroxime, cephalothin, streptomycin, tobramycin, tetracycline, oxytetracycline, erythromycin ethylsuccinate, clarithromycin, acetylspiramycin, azithromycin, roxithromycin, erythromycin, clindamycin and lincomycin. Further, the antibacterial substance added in the menstrual blood preservative fluid is preferably one or more of amphotericin, penicillin and streptomycin.
The method for washing the bottom precipitate comprises the following steps: washing with 10 times of buffer solution, centrifuging at 1000-1500rpm for 5-10 min, and discarding waste liquid. This process was repeated 3 times to remove residual bacteria, fungi and mycoplasma.
The adherent culture comprises primary culture and subculture amplification of the uterine membrane stem cells.
The primary culture method of the uterine membrane stem cells comprises the following steps: the target cells in the bottom pellet obtained after separation and washing were resuspended in 10ml of α -MEM (containing 10% -20% serum), and placed in a dish at 37 ℃ with 5% CO2After 24 hours, the cells were washed 3 times with 10ml of the buffer solution, and 10ml of the fresh culture solution was replaced, and then the culture solution was replaced every 3 days. When the cells grew to about 80% -90%, they were digested and then passaged.
The step of subculturing and expanding the endometrium stem cells comprises the steps of completely replacing the liquid of the endometrium stem cells after subculturing in a primary culture method every two days, digesting when the cells grow to about 80-90%, and continuously subculturing.
The invention also provides a high-efficiency uterine membrane stem cell banking method, which comprises the following steps: collecting menstrual blood, separating and sterilizing uterine membrane stem cells, amplifying, analyzing and detecting, storing for a long time and detecting regularly.
The collecting of menstrual blood, the separation and sterilization of the uterine membrane stem cells and the amplification in the library building method are the same as those in the separation and extraction method of the uterine membrane stem cells.
The analytical tests in the library construction method of the present invention include sterility testing, flow cytometry testing, karyotype testing, and differentiation capacity testing.
Long-term preservation of uterine membrane stem cells in the banking method of the present invention comprises subjecting the cells to a 1X 10 cell size6To 1X 107The density of the cells/ml is resuspended in a cell freezing medium containing cell culture solution, DMSO and serum, and is preserved by a programmed cooling method in a programmed cooling box in a refrigerator at minus 80 ℃ overnight, and then is transferred to liquid nitrogen at minus 196 ℃ for long-term preservation.
In the long-term storage process, the regular detection of the uterine membrane stem cells is carried out, and the regular detection comprises cell activity detection, sterility detection, flow cytometry detection and differentiation capacity detection after cell recovery.
Wherein the cell activity detection is performed once every 12 months, and the cell viability is detected by trypan blue staining after the cells are recovered.
Flow cytometry detection includes detection of cell surface antigens CD14, CD29, CD44, CD45, CD90, HLA.
The differentiation capacity test includes the tests of osteogenic differentiation capacity, adipogenic differentiation capacity and chondrogenic differentiation capacity.
In the method for establishing the bank of the uterine membrane stem cells, aseptic detection is carried out after amplification and regular detection in the freezing storage process, wherein the aseptic detection comprises detection of fungi and detection of bacteria.
One specific implementation mode of the uterine membrane stem cell separation and extraction method and the uterine membrane stem cell library building method comprises the following steps of:
1. collection of menstrual blood
Collecting menstrual blood with a menstrual cup, storing the menstrual blood in a menstrual blood storage solution, and transporting the obtained menstrual blood sample to a GMP workshop at 2-8 ℃, wherein the menstrual blood storage solution is PBS salt buffer solution or SPB salt buffer solution containing heparin sodium. In addition, antibacterial substance can be added into the menstrual blood preserving fluid.
2. Isolation and degerming of endometrial stem cells
Slowly adding an equal volume of menstrual blood sample into the human lymphocyte separation solution, centrifuging at the temperature of 15-25 ℃ and the rotating speed of 300-500g for 20-40 minutes; then collecting the bottom sediment, and washing the sediment to obtain stem cells in the endometrial tissue.
3. Primary culture of endometrial stem cells
Culturing the uterine membrane stem cells obtained by separation in a cell culture solution, and replacing a fresh culture solution after 24 hours. The number and morphology of stem cells isolated from the bottom pellet were observed by taking 40-fold and 100-fold photographs under a microscope. The solution was changed every 3 days later, and the cell morphology was recorded by photographing every day. When the cells grew to about 80% -90%, the numbers of the cells compared with the primary harvest were digested and passaged further. The culture solution is alpha-MEM containing 10% -20% serum.
4. Passage expansion of uterine membrane stem cells
After passage, the liquid of the uterine membrane stem cells is completely changed every two days, when the cells grow to be about 80% -90%, the number of the cells obtained by P1 generation is digested and contrasted, and passage is continued. The culture medium used in this step was α -MEM (containing 10% serum).
5. Detection of endometrial stem cells
And (3) carrying out a series of tests such as sterility test, flow cytometry test, karyotype test, osteogenic differentiation test and the like on the subcultured cells.
6. Cryopreservation of endometrial stem cells
Storing the uterus membrane stem cells after passage by a programmed cooling method, namely, the cells are stored at 1 × 106-To 1X 107The cells/ml density was resuspended in cell culture, DMSO and serum containing cell culture medium, stored overnight in a-80 ℃ freezer using a programmed cooling box, and then transferred to-196 ℃ liquid nitrogen for long term storage.
7. Periodic detection of endometrial stem cells
The uterine membrane stem cells are revived at regular intervals (for example, 12 months), and the quality of the preserved uterine membrane stem cells is monitored by detecting the cell viability, the sterility, the flow cytometer, the differentiation capacity and the like.
In this context, a range of values from one value to another is a general expression avoiding any recitation of all values in the range in the specification. Thus, recitation of a range of values herein is intended to encompass any value within the range and any smaller range defined by any value within the range, as if the range and smaller range were explicitly recited in the specification.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In practice, the invention will be understood to cover all modifications and variations of this invention provided they come within the scope of the appended claims.
Examples
The embodiment provides a specific method for separating, extracting and banking the uteromembranous stem cells, and the method also comprises the step of separating and extracting the stem cells by taking a traditional method for separating and extracting the uteromembranous stem cells as a comparative example.
The embodiment comprises the following steps:
step 1, collecting menstrual blood
Menstrual blood was collected on the second or third day of the menstrual cycle after the donor signed an informed consent, and 5ml to 15ml of the menstrual blood collected in a menstrual cup was stored in an equal volume of menstrual blood preservative solution (PBS salt buffer solution containing heparin sodium, and added with 100U/ml penicillin and 100U/ml streptomycin) and transported to a national-certified GMP workshop at 2 to 8 ℃ within 48 hours.
Comparative example: traditional separation and extraction of uterus membrane stem cells
Slowly adding 5ml of menstrual blood mixed solution into a 15ml centrifuge tube containing 5ml of Ficoll separating medium, utilizing a density gradient centrifugation method, setting the acceleration and the deceleration as 1, setting the rotating speed as 300-. The density gradient centrifugation photograph is shown in FIG. 1.
Wherein, the tunica albuginea layer can harvest mononuclear cells, thereby harvesting single tunica uteri stem cells; the endometrial tissue mass can be harvested by bottom sedimentation.
PBMC were washed with 10ml PBS, centrifuged at 1000-1500rpm for 5-10 min, and the waste stream discarded. This process was repeated 3 times to remove residual bacteria and the like.
The separated target cells were resuspended in 3ml of α -MEM (containing 10% serum), and placed in a six-well plate at 37 ℃ with 5% CO2After 24 hours, the cells were washed 3 times with 10ml of the buffer solution, and 3ml of the fresh culture solution was replaced, and the adherent cells were observed under a microscope to show a fusiform shape, and the cell photographs are shown in FIG. 2(a) and FIG. 2 (b). The sterility of the discarded supernatant was checked.
In this embodiment: efficient separation and extraction of endometrial stem cells
Slowly adding 5ml of menstrual blood mixed solution into a 15ml centrifuge tube containing 5ml of Ficoll separating medium, utilizing a density gradient centrifugation method, setting the acceleration and the deceleration as 1, setting the rotating speed as 300-500 Xg, centrifuging for 20-40 minutes, and collecting waste bottom sediment, wherein the bottom sediment contains a endometrial tissue block. The bottom pellet was washed with 10ml PBS, centrifuged at 1000-1500rpm for 5-10 minutes, and the waste was discarded. This process was repeated 3 times to remove residual bacteria and the like.
The separated target cells were resuspended in 10ml of α -MEM (containing 10% serum), and placed in a dish (6 times the surface area of a six-well plate) at 37 ℃ with 5% CO2After 24 hours, the cells were washed 3 times with 10ml of the buffer solution, 10ml of the fresh culture solution was replaced, and adherent cells were observed under a microscope and were in a clonal spindle shape, and the cell photographs are shown in FIG. 3(a) and FIG. 3 (b). The sterility of the discarded supernatant was checked.
Comparative example: primary culture and passage of uterine membrane stem cells
The six well plate of the comparative example of step 2 was replaced every 3 days, the cells grew to about 80% -90% for about 8 days, washed 3 times with 1ml of PBS, digested at 37 ℃ for 3-5 minutes, centrifuged to remove pancreatin, and resuspended in a plate with 10ml of α -MEM (containing 10% serum). And then, the liquid is completely changed every two days, when the cells grow to be full of about 80% -90%, the cells are digested and continuously passaged. FIGS. 4(a) and 4(b), and FIGS. 5(a) and 5(b) show photographs (8 days) of primary cells of comparative examples and this example. FIGS. 6(a) and 6(b), and FIGS. 7(a) and 7(b) are photographs of cells at P1 generation (13 days) and P1 generation (11 days), respectively, of comparative example and this example.
In this embodiment: primary culture of uterine membrane stem cells obtained by efficient separation and extraction
The plate of step 2 was replaced every 3 days, cells grew around 80% -90% around 8 days, washed 3 times with 5ml PBS, digested at 37 deg.C for 3-5 minutes, centrifuged to remove pancreatin, resuspended in plate with 10ml α -MEM (containing 10% serum) at 37 deg.C with 5% CO2Standing the mixture under the condition of (1) and carrying out subculture. And then, the liquid is completely changed every two days, when the cells grow to be full of about 80% -90%, the cells are digested and continuously passaged. FIGS. 5(a) and 5(b) show photographs (8 days) of primary cells of this example.
The number of the utero-membrane stem cells harvested by the conventional method (comparative example) and the efficient method of the present invention (example) primary culture and P1 subculture was counted and listed in table 1 below.
TABLE 1
As can be seen from Table 1 above, the number of uterine membrane stem cells harvested by the efficient method of the present invention is about 6.5-10 times that of the conventional method.
Step 4, detecting the uterine membrane stem cells
1) Sterility testing
And (3) sending the amplified uterine membrane stem cells into a standard laboratory of non-clinical research quality management standards for medicine quality detection, including sterility detection, mycoplasma detection and virus detection (detecting whether the isolated uterine membrane stem cells are polluted by bacteria or fungi according to a method of sterility detection in appendix XIIA of three parts of Chinese pharmacopoeia 2010).
2) Flow cytometry detection
Taking MenSCs of 3 rd generation in logarithmic phase, digesting with 0.25% trypsin-EDTA, neutralizing with alpha-MEM containing 10% -20% fetal calf serum, centrifuging at 1000-. Adjusting cell density to 106Each cell/ml, in a volume of 1ml, was dispensed into sterile centrifuge tubes. Mouse anti-human CD14-FITC, CD44-FITC, CD45-FITC, CD29-PE, CD90-PE and HLA-PE antibodies are added according to the amount of the specification respectively, mixed evenly and incubated for 30min at 4 ℃ in dark. Meanwhile, cells without added antibody are used as a negative control, and corresponding mouse anti-human IgG1 is used as an isotype control. After the incubation is finished, PBS is washed for 3 times to remove the unbound antibody, a BD flow cytometer detects the expression condition of the cell surface antigen, and CellQuest software processes the result.
The treatment results show that the CD14, CD29 and CD90 are positive, and the CD14, CD45 and HLA are negative.
3) Osteogenic differentiation assay
The cells were digested with 0.25% pancreatin-EDTA and treated with 10%4The cells/ml density was inoculated into 3 six-well plates, respectively, and induced to differentiate for 28 days after the cells were attached to the wall by replacing osteogenic induction liquid (alpha-MEM supplemented with 10% FBS, 100nM dexamethasone, 10mM sodium beta-glycerophosphate, and 0.05mM ascorbic acid). The osteogenesis inducing solution was replaced 2 times per week. After which the following tests were carried out.
3.1 alkaline phosphatase (ALP) assay
The activity of ALP was measured 14 days after the induction of differentiation with osteogenic induction liquid by the digestion of MenSCs: the culture medium in the six well plates was aspirated, washed 3 times with PBS, and 400. mu.l lysis buffer (1.5M Tris-HCl, 1mM ZnCl) was added2，1mM MgCl21% Triton-X100, pH 9.2), lysis at 37 ℃ for 30min, centrifugation at 13,000rpm for 10min, taking 20. mu.l of supernatant, detecting alkaline phosphatase activity with an ALP kit, and reading the absorbance at 405nm with an enzyme reader.
The calculated ALP detection value is 10IU/106And (4) cells.
3.2 alizarin Red mineralized node staining
When MenSCs cells were subjected to osteogenic induction and differentiation for 28 days, alizarin red staining was performed to observe the formation of mineralized nodules. The method comprises the following specific steps: the cells in the six-well plate were washed three times with PBS, 70% cold ethanol was added, fixed at 4 ℃ for 1 hour, washed 3 times with deionized water to remove residual ethanol, 3ml of 0.1% alizarin red solution (pH 8.3) was added, stained at room temperature for 15min, shaken intermittently, washed 5 times with deionized water to remove unbound alizarin red, washed 37 ℃ with PBS for 15 min. The red mineralized nodules were observed by microscopic observation.
When the cells of this example are grown to about 80% -90%, they are digested with pancreatin (containing EDTA), washed 3 times with PBS, and sent to a laboratory of standard quality management standards for non-clinical research of pharmaceuticals for quality testing, including sterility testing, mycoplasma testing, cell counting and viability testing, to determine whether they reach the acceptable standards. The detected standard uterine membrane stem cells are expressed by 1 × 106～2×107The density of individual cells/ml was resuspended in a cryopreservation solution (DMSO, fetal bovine serum and α -MEM medium 1:2:7) and dispensed into cryopreservation tubes. Labeling outside the cryopreservation pipe, recording detailed information of cells on the label, including supplier name, cell algebra, cell culture solution components and concentration, cell inoculation density, cryopreservation solution components, cryopreservation date and the like, completely recording the real freezing condition of each sample, accurately scanning the information into a data management system, and completing cell library information entry and construction. Placing the freezing tube into a programmed cooling box, standing overnight in a refrigerator at-80 deg.C, and transferring the freezing tube into a corresponding space in a liquid nitrogen tank at-196 deg.C for long-term storage the next day. The freezing and storing space has good sanitary environment, including clean area, ventilation and lighting environment, and meets the relevant national file regulation.
And (3) resuscitating a part of the cryopreserved uterine membrane stem cells every 12 months, taking 50 mul of cell suspension, adding 50 mul of trypan blue staining solution, staining for 3min, adding the cell mixed solution into a cell counting plate, observing under a microscope, and calculating the proportion of cells which are not stained into blue, namely the cell survival rate, wherein the cell survival rate is more than or equal to 95%. And performing various detections such as sterility detection, flow cytometry detection, osteogenic differentiation detection and the like according to the method so as to monitor the quality of the preserved uterine membrane stem cells. Test reports are periodically given to the donor.
The inventor of the invention provides a high-efficiency method for separating, extracting and establishing the uterine membrane stem cells through research and development, and after menstrual blood samples are obtained, waste bottom sediment in the traditional method is washed through density gradient centrifugation and then is cultured in an adherent manner to obtain a large amount of uterine membrane stem cells. A large amount of uterine membrane stem cells with higher purity can be obtained through amplification culture, and the uterine membrane stem cells are preserved for a long time and periodically subjected to quality detection, so that a large amount of uterine membrane stem cell resources are provided for future clinical and scientific research application and research.
In light of the above teachings, those skilled in the art will readily appreciate that the materials and their equivalents, the processes and their equivalents, as listed or exemplified herein, are capable of performing the invention in any of its several forms, and that the upper and lower limits of the parameters of the materials and processes, and the ranges of values between these limits are not specifically enumerated herein.
Claims (9)
1. A separation and extraction method of uterine membrane stem cells is characterized in that menstrual blood samples are subjected to density gradient centrifugation to obtain bottom sediment, the sediment is washed and then cultured in an adherent manner, and uterine membrane stem cells are harvested; wherein the content of the first and second substances,
the step of performing density gradient centrifugation and obtaining a bottom precipitate comprises:
collecting waste bottom sediment after density gradient centrifugation, wherein the bottom sediment contains endometrial tissue blocks, and washing the bottom sediment; the density gradient centrifugation is to slowly add an equal volume of menstrual blood sample on the human lymphocyte separation liquid, centrifuge for 20-40 minutes at the temperature of 15-25 ℃ and the rotating speed of 300-500 g;
the adherent culture comprises primary culture, and the method for the primary culture comprises the following steps: the target cells obtained after washing were resuspended in α -MEM and plated at 37 ℃ with 5% CO2After static culture under the condition of (1), washing with a buffer solution, and replacing a fresh culture solution; mixing the aboveThe plate in the step is changed with liquid once every 3 days; digesting when the cells are full of about 80% -90%, removing pancreatin after centrifugation, then resuspending in a plate at 37 deg.C and 5% CO2Standing for passage under the condition of (1).
2. The method for separating and extracting the uterine membrane stem cells as claimed in claim 1, wherein in the primary culture method, the target cells are resuspended in α -MEM and placed in a dish at 37 ℃ and 5% CO2The time of the stationary culture under the conditions of (1) is 24 hours, the α -MEM used is an α -MEM containing 10% to 20% serum, the amount of the α -MEM used is 10ml, the buffer used for washing is 10ml buffer, the number of washing is three, and the replacement of a fresh culture medium means the replacement of 10ml of a fresh culture medium.
3. The method for separating and extracting the uterine membrane stem cells as set forth in claim 1, wherein the washing of the bottom precipitate is performed by washing with 10 times the volume of the buffer solution, and the washing is repeated three times.
4. The method for separating and extracting uterine membrane stem cells according to claim 1, wherein the menstrual blood sample is menstrual blood of the second day or the third day of the female menstrual cycle, and an equal volume of buffer containing one or more antibacterial substances is added.
5. The method for separating and extracting uteromembrane stem cells according to claim 4, wherein the antibacterial substance is selected from one or more of amphotericin, penicillin, cephradine, cefazolin, cefadroxil, cefuroxime, cephalothin, streptomycin, tobramycin, tetracycline, oxytetracycline, erythromycin ethylsuccinate, clarithromycin, acetylspiramycin, azithromycin, roxithromycin, erythromycin, clindamycin and lincomycin.
6. The method for separating and extracting the uteromembrane stem cells according to claim 1, wherein the adherent culture comprises passage expansion.
7. The method for separating and extracting the endometrium stem cells according to claim 6, wherein the step of subculturing and amplifying the utero membrane stem cells comprises the following steps: completely replacing the fluid once every two days for the uteromembranous stem cells after passage in the primary culture method, digesting when the cells grow to be about 80-90%, and continuously performing passage; the culture medium used in this step was α -MEM containing 10% serum.
8. A method for banking uteromembrane stem cells, which is characterized by comprising the steps of menstrual blood collection, separation and extraction of uteromembrane stem cells, primary culture, subculture, amplification, analysis and detection, long-term storage and periodic detection, wherein the method of any one of claims 1-7 is adopted for the menstrual blood collection, separation, sterilization and extraction of uteromembrane stem cells, primary culture and amplification.
9. The method for banking uterine membrane stem cells according to claim 8, wherein the analytical tests comprise a sterility test, a flow cytometer test, a karyotype test, and a differentiation capacity test; the items for periodic detection comprise cell activity detection, sterility detection, flow cytometer detection and differentiation capacity detection after cell recovery.
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