CN115651897A - Kit for separating, purifying and amplifying endometrial mesenchymal stem cells and application thereof - Google Patents
Kit for separating, purifying and amplifying endometrial mesenchymal stem cells and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a kit for quickly and efficiently separating, purifying and amplifying endometrial mesenchymal stem cells and application thereof. The kit consists of endometrial tissue digestive juice, an endometrium mesenchymal cell culture reagent and an endometrium mesenchymal stem cell culture reagent. The endometrial tissue digestive juice comprises: collagenase type I, collagenase type II, collagenase type IV, DNAse I and DMEM/F12 basal medium. The endometrium interstitial cell culture reagent comprises: fetal bovine serum, TGF-beta type I receptor inhibitor, antibiotics and DMEM/F12 basal medium. The culture reagent for the endometrial mesenchymal stem cells comprises: the mesenchymal stem cells are fetal bovine serum, TGF-beta type I receptor inhibitor, antibiotics and DMEM/F12 basal medium. The invention mainly adds TGF-beta I type receptor inhibitor in the endometrium mesenchymal cell culture stage and the endometrium mesenchymal stem cell amplification culture stage respectively, so that the separation, purification and amplification efficiency of the endometrium mesenchymal stem cell is greatly improved.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for quickly and efficiently separating, purifying and amplifying endometrial mesenchymal stem cells and application thereof.
Background
Human endometrium is a highly dynamic tissue with the ability to regenerate cyclically during reproductive years, which is closely related to the presence of endometrial stem cells (EnSCs) in endometrial tissue. It was found that the EnSC mainly consists of a group of epithelial stem cells (EpSCs), endometrial mesenchymal stem cells (eMSCs) and Side Population of Stem Cells (SPSCs). The eMSC serving as a seed cell ideal for regenerative medicine has the characteristics of simple acquisition and secretion of various relevant factors such as promotion of cell proliferation and angiogenesis, and has the potential effects of promoting endometrial growth and recovering female reproductive function. Creates better precondition for the treatment of diseases by stem cell transplantation, especially for the treatment of diseases in the female reproductive field. Besides, eMSCs can be used as immunomodulators to relieve inflammation, influence angiogenesis in tissue regeneration and induce pluripotent stem cells; eMSCs have the ability to differentiate into smooth muscle cells and have been attempted for use in the reconstruction of the bladder wall. In addition, it has been studied that embcs and bone marrow derived mesenchymal stem cells (BMSCs) are transplanted into a myocardial-infarcted heart, respectively, and it has been observed that the infarcted area of the heart into which the embcs were transplanted is smaller. The data show that eMSCs has greater superiority and potential in clinical application than other stem cells.
eMSCs, as an attractive source of stem cell regenerative therapy, have the major advantages of being non-invasively available (via non-invasive biopsy of endometrial tissue or collection of menstrual blood) and significantly differentiating in vitro, being a very stable source of autologous stem cells, and being more readily expanded in vitro than stem cells from other sources. Studies have shown that eMSCs are distributed mainly around the blood vessels of the endometrial function layer and basal layer, and the Sushi domain stabilizing 2 (SUSD 2) protein around the blood vessels can serve as a molecular marker for eMSCs identification. At present, eMSC separation and purification modes mainly comprise: clone purification and flow cytometric sorting or magnetic bead sorting based on the SUSD2 protein tag. Specifically, the cloning and purification method comprises the steps of firstly separating endometrial stromal cells (including endometrial stromal cells and endometrial mesenchymal stem cells) by a method combining mechanical shearing and collagenase digestion, and then further purifying eMSC by using a monoclonal loop method. The SUSD2 protein tag-based flow cytometry sorting or magnetic bead sorting is still the mainstream separation method of the eMSC at present, the method has the obvious advantage of high purity of the obtained eMSC, the whole preparation period is shorter than that of a cloning purification method, but the 1 × 10 can be obtained by still needing about 20-30 days 6 eMSCs (slightly different time required for different individual sources).
Generally, the existing eMSC separation, purification and amplification efficiency is low, the objective problem is still one of the technical bottlenecks limiting the large-scale development of clinical application of eMSC, how to efficiently separate, purify and amplify eMSC becomes the focus of attention in the field, and the establishment of an efficient eMSC separation/amplification culture system is urgent and has important clinical significance. If the existing eMSC separation steps/means, culture or amplification systems can be optimized and the kit for efficient separation, purification and amplification of eMSC is developed, the eMSC is promoted to be converted from basic research to clinical application, and meanwhile, the current situation of the regenerative medicine industry developed based on eMSC is also promoted powerfully.
Disclosure of Invention
Solves the technical problem
The invention aims to provide a culture system for efficiently separating, purifying and amplifying eMSC, and the culture system is prepared into a kit and is particularly applied to the fields of basic scientific research and clinical transformation medicine.
Adopts the technical scheme
The kit for separating, purifying and amplifying the endometrial mesenchymal stem cells is characterized by comprising endometrial tissue digestive juice, an endometrial mesenchymal cell culture reagent and an endometrial mesenchymal stem cell culture reagent.
Further, the endometrial tissue digestion fluid comprises: collagenase type I, collagenase type II, collagenase type IV, DNAse I and DMEM/F12 basal medium.
Further, the endometrial stromal cell culture reagent comprises: fetal bovine serum, TGF-beta type I receptor inhibitor, antibiotics and DMEM/F12 basal medium.
Further, the endometrium mesenchymal stem cell culture reagent comprises: the mesenchymal stem cells are fetal bovine serum, TGF-beta type I receptor inhibitor, antibiotics and DMEM/F12 basal medium.
Furthermore, the kit for efficiently separating, purifying and amplifying the endometrial mesenchymal stem cells is applied to basic research and clinical transformation medicine.
Specifically, the method comprises the following steps:
firstly, dissociating endometrial tissue into single cell suspension by adopting a method of combining mechanical shearing and mixed collagenase digestion, sequentially passing through a 100/200/400-mesh screen, collecting filtrate and centrifuging, then removing supernatant, culturing by using a DMEM/F12 culture medium containing fetal calf serum, a TGF-beta I type receptor inhibitor and antibiotics, sorting magnetic beads by using a SUSD2 antibody after cells grow and converge, and performing amplification culture on the sorted SUSD2 positive cells by using the DMEM/F12 culture medium containing fetal calf serum for mesenchymal stem cells, the TGF-beta I type receptor inhibitor and the antibiotics.
The eMSC separation, purification and amplification kit comprises endometrial tissue digestive juice, an endometrial mesenchymal cell culture reagent and an endometrial mesenchymal stem cell culture reagent. The endometrial tissue digestive juice comprises: collagenase type I, collagenase type II, collagenase type IV, DNAse I and DMEM/F12 basal medium; the endometrium interstitial cell culture reagent comprises: fetal bovine serum, TGF-beta type I receptor inhibitor, antibiotics and DMEM/F12 basal medium; the culture reagent for the endometrial mesenchymal stem cells comprises: the mesenchymal stem cells are fetal bovine serum, TGF-beta type I receptor inhibitor, antibiotics and DMEM/F12 basal medium.
Advantageous effects
The eMSC separation, purification and amplification kit provided by the invention has the advantages that:
the eMSC separation, purification and amplification kit provided by the invention is different from the traditional eMSC preparation method, and mainly adds TGF-beta I type receptor inhibitors in the endometrium interstitial cell culture stage and the eMSC amplification culture stage after magnetic bead sorting, so that the eMSC separation, purification and amplification efficiency is greatly improved. The successful development of the kit provides great convenience for the development of the relevant basic scientific research around eMSC, and is more beneficial to the development of the clinical research and the transformation application of eMSC.
Drawings
FIG. 1 Primary cultures were observed in brightfield after days 4, 8 and 12 and photographed.
FIG. 2 Crystal Violet staining after day 12 of primary culture to analyze cell colony formation.
FIG. 3 enrichment ratio analysis of SUSD2+ eMSCs after 12 days of primary culture.
FIG. 4 analysis of the cell surface marker expression after the magnetic bead sorting SUSD2+ eMSC amplification culture.
FIG. 5 is a schematic flow chart of the present invention.
Detailed Description
The overall principle and improvement point of the embodiment of the invention are shown in fig. 5.
1. Endometrial tissue specimen inclusion standard
(1) Selecting patients who are clinically resected in the whole uterus due to the myoma of the uterus between muscle walls or cervical lesion affecting the uterus
(2) The patient is not treated by hormone or other medicines before operation;
(3) The patient does not have other gynecological reproductive endocrine diseases and malignant tumor diseases;
2. primary culture of endometrioid cells
(1) Fresh endometrial tissue obtained by surgery is placed into a 50mL centrifuge tube containing a pre-cooled PBS solution and transported to the laboratory within 30 minutes at low temperature.
(2) The tissue was removed from the cell culture dish and rinsed 2-3 times with PBS containing normal double antibody concentrations, sterile forceps transferred the tissue to a 1.5mL centrifuge tube, the tissue was cut into 1mm3 pieces with sterile fine-head surgical scissors, the cut pieces were transferred to a 15mL centrifuge tube with a 1mL pipette, and 4 volumes of endometrial tissue digest were added from DMEM/F12 (Biosharp, BL 305A) media containing 0.5MG/mL collagenase type I (Biosharp, BS 163), 0.5MG/mL collagenase type II (Biosharp, BS 164), 0.5MG/mL collagenase type IV (Biosharp, BS 165) and 1U/μ L DNAse I (BioFroxx, 1121 MG), digested in a water bath at 37 ℃ for 40 minutes, during which the tube was inverted repeatedly to expose the tissue to the digest solution at full contact with the tissue at intervals of 10 minutes until the pieces were fully dispersed into a cell suspension 010.
(3) Sequentially sieving the cell suspension with 100 mesh, 200 mesh and 400 mesh cell sieves, collecting the cell suspension after sieving, centrifuging at 300g for 5min, discarding the supernatant, retaining the cell precipitate, centrifuging at 300g for 5min, washing twice with PBS, discarding the supernatant, retaining the cell precipitateStarch, resuspending the cell pellet with endometrium stromal cell culture reagent consisting of 10% FBS (ExCelBio, FSP 500), 1 XPen-Strep-Ampho. BSolution (Biological indicator, 03-033-1B), 2mM GlutaMax, and plating onto 100mM cell culture dish TM (Gibico, 35050-061) and 1nM A83-01 (MCE, HY-10432, inhibitor of TGF-. Beta.type I receptor). The culture medium is completely discarded the next day and the culture dish is rinsed twice with PBS, the dead cells which are not attached to the wall are cleaned, then fresh endometrium interstitial cell culture reagent (the specific components are the same as those described above) is added, and the subsequent magnetic bead sorting experiment is carried out when the cells grow to 80% confluence.
3. Magnetic bead sorting SUSD2+ eMSCs
3.1 cell sample preparation
(1) Taking out the primary cultured endometrial stromal cells, removing the culture medium, and washing with PBS for 2 times;
(2) Digesting 0.25% Trypsin (Biosharp, BL 512A) for 10min;
(3) Resuspending the endometrium interstitial cell culture reagent, repeatedly blowing and beating the reagent, and transferring the reagent into a 15mL centrifuge tube;
(4) Centrifuging for 3min at 300g, and discarding the supernatant;
(5) MACS Buffer (Miltenyi Biotec, 130-091-221) 1 mL/tube was added, resuspended, and cells counted;
(6) Centrifuge at 300g for 10min and discard the supernatant.
3.2 magnetic labeling of cells
(1) At 95. Mu.L/10 6 Adding MACS Buffer into each cell, and resuspending;
(2) At a ratio of 5. Mu.L/10 6 Individual cells were added to PE anti-human SUSD2 antibody (BioLegend, 327406);
(3) Keeping out of the sun, and incubating for 15min at 4 deg.C in a refrigerator;
(4) Adding MACS Buffer to 4mL, and washing;
(5) Centrifuging at 300g for 10min, and discarding the supernatant;
(6) At 90 μ L/10 6 Adding MACS Buffer to each cell for resuspension;
(7) At a ratio of 10. Mu.L/10 6 Adding Anti-PE MicroBeads (Miltenyi Biotec, 130-048-801) to each cell;
(8) Placing the cell suspension in a refrigerator at 4 deg.C, and incubating for 15min in dark;
(9) 1mL MACS Buffer;
(10) Centrifuging at 300g for 10min, and discarding the supernatant;
(11) 1mL of MACS Buffer was resuspended.
3.3 magnetic sorting SUSD2+ eMSCs
(1) Mounting an MS sort column (Miltenyi Biotec, 130-122-727) on a MACS separator;
(2) Slowly adding 500 mu L of MACS Buffer into the MS sorting column for rinsing for 1 time to avoid generating bubbles;
(3) When the liquid in the MS sorting column is drained, adding cell suspension into the sorting column by times according to 500 mu L/time;
(4) Slowly adding MACS Buffer into the MS sorting column according to 500 mu L/time for 3 times, and washing the cells which are not combined with the magnetic beads;
(5) Taking down the MS sorting column, and placing the MS sorting column on a new 15mL centrifuge tube;
(6) Adding 1mL of MACS Buffer into the MS sorting column, uniformly and forcibly pressing down the piston, and collecting SUSD2+ eMSC suspension flowing out of the lower end of the sorting column;
(7) The SUSD2+ eMSCs suspension was centrifuged at 300g for 5min, the supernatant was discarded to retain the cell pellet, followed by 1 PBS wash and the supernatant was discarded to retain the cell pellet.
(8) And re-suspending the reagent into a single cell suspension by using the endometrial mesenchymal stem cell culture reagent, and paving the suspension into a cell culture dish for amplification culture. The medium consists of 10% FBS (Viva cell, C04400-500), 1 XPen-Strep-Ampho. B Solution, 2mM GlutaMax TM And 1nM of A8301 in DMEM/F12.
(9) After the cells are full of the above (8), the ratio of 1: and 3, continuously using the endometrial mesenchymal stem cell culture reagent for amplification culture.
The following is a further description of the invention:
collecting endometrial tissue meeting the inclusion standard to perform separation, purification and amplification tests of endometrial mesenchymal stem cells, preparing single cell suspension by adopting a method combining mechanical shearing and mixed collagenase digestion, and dividing the single cell suspension into one partTo do two, the cell pellet was retained by discarding the supernatant after centrifugation, and the endometrial stromal cell culture reagent (containing 10% FBS, 1 XPen-Strep-Ampho. B Solution, 2mM GlutaMax) without A83-01 and containing A83-01 was used, respectively TM DMEM/F12 medium) were resuspended into single cell suspensions and plated on 6-well plates, and the formation of monoclonal colonies using the a8301 group in the primary culture medium was observed by bright field photography after days 4, 8, and 12 of the primary culture, which was better than that using the a8301 group in the primary culture medium (see fig. 1).
Subsequently, we performed crystal violet staining experiments on primary culture cell clones at day 12 of primary culture, counted the number of cell clones in each of 3 wells, and confirmed that: the clone number of the group A83-01 added with the endometrium interstitial cell culture reagent is obviously higher than that of the group A83-01 not added with the endometrium interstitial cell culture reagent (see table 1 and figure 2).
TABLE 1 analysis of cell colony formation by Crystal Violet staining after day 12 of Primary culture
Meanwhile, the expression of SUSD2 molecules on the surface of the endometrium mesenchymal stem cells is analyzed in a flow mode after cell digestion on the 12 th day of primary culture by using different culture mediums, and the result shows that the SUSD2 positive cell proportion is 39.3% when A83-01 is added to the endometrium mesenchymal cell culture reagent, and the positive cell proportion is 3.84% when A83-01 is not added to the endometrium mesenchymal cell culture reagent (see table 2 and figure 3), namely the enrichment proportion of SUSD2 positive cells (namely eMSC) in the primary cells is improved by 10.23 times by adding A83-01 to the endometrium mesenchymal cell culture reagent.
TABLE 2 enrichment ratio analysis of SUSD2+ eMSCs after 12 days of primary culture
Furthermore, we also analyzed the SUSD2 positive cells (i.e., eMSCs) after magnetic bead sorting by flow cytometry for other surface marker expression, and the results showed that: compared with the method without adding A8301, the stem cell markers (including CD73, CD90 and CD 105) on the surface of eMSCs and the non-stem cell markers (CD 34, CD45 and HLA-DR) obtained by adding A83-01 in the separation, purification and amplification processes of eMSCs have consistent expression (see table 3 and figure 4), which indicates that the addition of A83-01 in the separation, purification and amplification processes of eMSCs can obviously improve the enrichment ratio of SUSD2 positive cells and does not influence the expression of the stem cell markers on the surface of eMSCs.
TABLE 3 analysis of marker expression on cell surface after magnetic bead sorting SUSD2+ eMSCs amplification culture
Note: CD73, CD90, CD105: mesenchymal stem cell surface marker; CD34: surface marker of hematopoietic stem cells; CD45: white blood cell surface marker; HLA-DR: t lymphocyte surface marker.
The kit provided by the invention is mainly characterized in that endometrial tissue digestive juice, an endometrial mesenchymal cell culture reagent and an endometrial mesenchymal stem cell culture reagent are optimized. Especially, the addition of TGF-beta type I receptor inhibitor (not limited to A83-01) in the endometrial mesenchymal cell culture reagent can obviously improve the enrichment ratio (up to 10 times) of the endometrial mesenchymal stem cells in primary endometrial mesenchymal cells. This lays a foundation for rapidly obtaining sufficient quantity of the endometrium mesenchymal stem cells. In a word, the kit provided by the invention can be used for rapidly separating, purifying and amplifying the endometrial mesenchymal stem cells, and promoting the basic research and clinical transformation application of the endometrial mesenchymal stem cells.
Claims (5)
1. The kit for separating, purifying and amplifying the endometrial mesenchymal stem cells is characterized by comprising endometrial tissue digestive juice, an endometrial mesenchymal cell culture reagent and an endometrial mesenchymal stem cell culture reagent.
2. The kit for isolating, purifying and amplifying the endometrial mesenchymal stem cells according to claim 1, wherein the endometrial tissue digestion solution comprises: collagenase type I, collagenase type II, collagenase type IV, DNAse I and DMEM/F12 basic culture medium.
3. The kit for isolating, purifying and amplifying the endometrial mesenchymal stem cells according to claim 2, wherein the endometrial mesenchymal cell culture reagent comprises: fetal bovine serum, TGF-beta type I receptor inhibitor, antibiotics and DMEM/F12 basal medium.
4. The kit for isolating, purifying and amplifying the endometrial mesenchymal stem cells according to claim 3, wherein the kit for culturing the endometrial mesenchymal stem cells comprises: the mesenchymal stem cells are fetal bovine serum, TGF-beta type I receptor inhibitor, antibiotics and DMEM/F12 basal medium.
5. Use of the kit for the isolation, purification and amplification of endometrial mesenchymal stem cells according to any one of claims 1 to 4 in basic research and clinical transformation medicine.
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Inventor after: Gui Tao Inventor after: Ma Jiehua Inventor after: Huang Meihua Inventor after: Cheng Chen Inventor after: Wu Wenjin Inventor after: Wang Jindan Inventor after: Wei Xinjun Inventor after: Wan Guiping Inventor after: Wang Zhihui Inventor after: Cao Meiling Inventor after: Cao Jian Inventor after: Xia Shuyu Inventor after: Gan Lin Inventor after: Chen Huixian Inventor after: Zhou Wenjuan Inventor before: Gui Tao Inventor before: Ma Jiehua Inventor before: Huang Meihua Inventor before: Cheng Chen Inventor before: Wu Wenjin Inventor before: Wang Jindan Inventor before: Wei Xinjun Inventor before: Wan Guiping Inventor before: Wang Zhihui Inventor before: Cao Meiling Inventor before: Cao Jian Inventor before: Xia Shuyu Inventor before: Gan Lin Inventor before: Chen Huixian Inventor before: Zhou Wenjuan |