CN117925518A

CN117925518A - Method for extracting endometrial mesenchymal stem cells in ectopic focus of adenomyosis

Info

Publication number: CN117925518A
Application number: CN202410175043.7A
Authority: CN
Inventors: 桂涛; 曹剑; 魏鑫俊; 仇影影; 马洁桦; 甘霖; 黄美华; 王智慧; 王金丹; 夏舒羽; 万贵平
Original assignee: Jiangsu Institute Of Traditional Chinese Medicine Jiangsu Integrated Traditional Chinese And Western Medicine Hospital
Current assignee: Jiangsu Institute Of Traditional Chinese Medicine Jiangsu Integrated Traditional Chinese And Western Medicine Hospital
Priority date: 2024-02-07
Filing date: 2024-02-07
Publication date: 2024-04-26

Abstract

The invention discloses a method for extracting endometrial mesenchymal stem cells from ectopic focus of adenomyosis. The invention takes an ectopic focus which is aseptically cut after the total hysterectomy of a patient with adenomyosis as a material, adopts a method combining an enzyme digestion method and a tissue block method to extract primary endometrial cells in the ectopic focus of adenomyosis, and then uses a PE marked SUSD2 antibody to separate endometrial mesenchymal stem cells according to a conventional magnetic bead separation method. By using the method, the endometrial mesenchymal stem cells of the ectopic focus of the adenomyosis can be extracted efficiently; the obtained endometrium mesenchymal stem cells can still maintain the form, surface molecular marker expression, in-vitro proliferation activity and directional differentiation potential of the mesenchymal stem cells after in-vitro multiple passage expansion. The extraction method has the most remarkable advantages of short extraction period, high separation success rate and simple operation technology, and can rapidly obtain a large number of ectopic endometrial mesenchymal stem cells.

Description

Method for extracting endometrial mesenchymal stem cells in ectopic focus of adenomyosis

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for efficiently extracting endometrial mesenchymal stem cells in adenomyosis ectopic disease and application thereof.

Background

Hysteromyopathy (Adenomyosis, AM) is a common and high incidence of women of childbearing age, and the menorrhagia, dysmenorrhea, infertility and the like caused by the hysteromyopathy seriously affect the physical and mental health of patients. Currently, conservative treatment regimens are generally not satisfactory except for hysterectomy. AM pathogenesis theory is numerous and mainly includes "endometrium basal portion invagination and tissue injury repair theory" and "mullerian duct remains metaplasia and adult stem cell differentiation theory". Among them, the adult stem cell differentiation theory considers that adenomyosis originates from embryonic multipotent stem cell metaplasia in the myometrium, including differentiation of endometrial epithelial progenitor cells and endometrial mesenchymal progenitor cells planted in the myometrium upon reverse flow of menstrual blood. In recent years, with the advent and application of single cell sequencing technology, multiple studies have demonstrated that pluripotent stem cells (Multipotential STEM CELLS) play an important role in the formation of ectopic foci, the exacerbation of pain, and the formation of fibrosis. Among them, SUSD2 expression positive endometrial mesenchymal stem cells (Endometrial MESENCHYMAL STEM CELLS, eMSCs) are particularly attracting attention and high importance in the art as one of the most important multipotent stem cells in ectopic lesions. There is evidence that endometrial mesenchymal stem cells in ectopic foci have stronger migration/invasion, anti-apoptotic capacity, differentiation potential, etc. than endometrial mesenchymal stem cells in normal endometrium, and these enhanced cell biological properties can be blocked by small molecule inhibitors. Thus, the development of related studies around endometriosis-derived mesenchymal stem cells would be one of the important paths for research of pathogenesis of adenomyosis and research of individual targeted therapies.

It is obvious that an important premise of developing relevant basic or application studies about ectopic focus of endometrial mesenchymal stem cells is that ectopic endometrial mesenchymal stem cells can be obtained relatively easily, and that the method for separating the mesenchymal stem cells from endometrium of uterine cavity is relatively mature and reliable through searching documents, but less reports are reported on the separation scheme for obtaining the ectopic endometrial mesenchymal stem cells from ectopic focus of myometrium of uterine adenomyosis. The existing primary separation method of the ectopic endometrium mesenchymal stem cells is single and mainly adopts an enzyme digestion method, but the method has the obvious defects of low culture success rate, low purity of the primary cells, long separation period and the like. The reason for this is mainly that the local microenvironment around the cells in the ectopic focus tissue is changed rapidly after the tissue mass is thoroughly dissociated by enzyme digestion, so that the activity and the adherence (adhesion) capability of the endometrial mesenchymal stem cells with extremely low content in the focus are greatly reduced, and the separation and the culture efficiency of the endometrial mesenchymal stem cells with ectopic focus are obviously reduced. The tissue block method is used as one of the classical primary tissue cell separation representative technologies, is very suitable for primary cell culture of soft tissues (such as skin, fat, umbilical cord and the like) and has relatively rich target cell content, and the technology has the advantages of simple operation, small influence on cell viability, low separation cost and the like. However, the ectopic focus tissue of the adenomyosis is characterized by hard texture, difficult accurate identification during the postoperative material taking, extremely low content of the endometrial mesenchymal stem cells and the like, and no research report on successfully culturing the ectopic endometrial mesenchymal stem cells by adopting a simple tissue mass method has been found yet. In conclusion, the traditional separation method of the endometrial mesenchymal stem cells in the ectopic focus of the adenomyosis has low efficiency and low success rate, and severely limits the development of the research direction of the endometriosis endometrial mesenchymal stem cells in the research field of the adenomyosis. Therefore, expanding a more effective extraction method of the endometriosis focus endometrium mesenchymal stem cells is a technical problem to be solved in the field.

According to the search literature, ma Yange et al found that in the article entitled "endometriosis and adenomyosis in situ, primary culture of ectopic endometrium interstitial cells and morphological observation" reported in volume 30 of China journal of women and children health care "2015, it was pointed out that ectopic endometrium interstitial cells (endometrial stroma cells, ESCs) can be successfully isolated and cultured by digesting and dissociating adenomyoma tissue of uterus for 3-5 hours in a constant temperature water bath box at 37 ℃ by adopting mixed enzyme liquid (0.5 mg/mL type IV collagenase and 0.125% trypsin), and the success rate of the separation and culture of the cells is 86.7%. However, the study was still complete dissociation of tissue by enzymatic digestion alone to release the cells within the tissue in order to purify the endometrial mesenchymal cells instead of the endometrial mesenchymal stem cells. Because the content of the endometrium mesenchymal stem cells in the ectopic focus tissue is extremely low, the single enzyme digestion for 3-5 hours greatly damages the cell activity of cell types with lower content (including the endometrium mesenchymal stem cells and the like), and finally the separation and culture efficiency of the endometrium mesenchymal stem cells in the ectopic focus is seriously reduced.

Disclosure of Invention

The invention aims to provide a method for efficiently obtaining endometrial mesenchymal stem cells in endometriosis ectopic focus and application of the mesenchymal stem cells in basic research and transformation medicine. The method can quickly and efficiently separate large-quantity and high-quality endometrial mesenchymal stem cells from the ectopic focus of the adenomyosis under the condition of lower cost, and can be applied to basic research related to pathogenesis of the adenomyosis and application research of personalized targeted therapy.

The aim of the invention is achieved by the following technical scheme:

A method for efficiently extracting endometrial mesenchymal stem cells in endometriosis lesions is characterized in that the method adopts a method of combining enzyme digestion and tissue mass for extracting the endometrial mesenchymal stem cells.

Wherein the enzymatic digestion process is: the tissue is placed on a rotary mixer arranged in a constant temperature incubator by adopting special compound enzyme digestive juice, and is subjected to digestion and dissociation in a low-speed 360-degree rotary mode.

1) In the enzyme digestion method, the complex enzyme digestion solution consists of trypsin with the final concentration of 1-1.5 mg/mL, collagenase type IV with the final concentration of 0.25-0.75 mg/mL and deoxyribonuclease with the final concentration of 0.5-1.5U/mu L. Preferably, the final concentration is 1.25 mg/mL trypsin and 0.5. 0.5 mg/mL type IV collagenase.

2) In the above-mentioned enzyme digestion method, the digestion time of the complex enzyme digestion solution should be controlled to be 15 to 45 minutes, and preferably, the digestion time of the complex enzyme is 30 minutes.

3) In the above-mentioned enzymatic digestion method, the digestion dissociation is carried out by using a low-speed 360 rotation, and the rotation speed is preferably controlled to be 0 to 20 rpm, and the rotation speed is preferably set to 10 rpm.

Wherein, the tissue block method is as follows: after the completion of the enzyme digestion, tissue mass treatment was performed. First, the digested tissue suspension is centrifuged, the supernatant is discarded and the tissue pellet is retained. Then, a small amount of fetal bovine serum was added to resuspend the tissue pellet and spread in a petri dish using a sterile pipette tip, transferred to an incubator and started to incubate upside down. Finally, when the tissue blocks cultivated in an inverted way are firmly attached, the cell culture dish is turned over, and a small amount of complete culture medium is added to perform the normal culture until the cells climb out.

1) In the above-mentioned tissue mass method, the tissue mass method needs to be carried out after the completion of the enzyme digestion method, and the time for carrying out the tissue mass method after the completion of the enzyme digestion should be controlled to be within 15 minutes, preferably, the tissue mass method should be carried out immediately after the completion of the enzyme digestion method, and the faster and better the tissue mass method should be.

2) The addition of a small amount of serum to resuspend the tissue and spread evenly in a 10 cm dish using a sterile pipette tip, and the subsequent transfer to the incubator should be kept for a resting time of between 1 and 5 minutes, preferably 3 minutes.

3) The inversion culture dish starts inversion culture, and the inversion culture time is controlled between 1 hour and 3 hours. Preferably, the inversion incubation time is 2 hours.

4) The addition of a small amount of complete medium is carried out in the forward culture until the cells climb out, the volume of complete medium added should be 2 to 6 mL, and preferably the volume of complete medium added is 4 mL.

The invention has the beneficial effects that: the method is suitable for extracting the mesenchymal stem cells from the ectopic focus of the adenomyosis, skillfully and organically combines enzyme digestion and tissue mass method according to the pathological characteristics of the ectopic focus of the adenomyosis, and has the remarkable advantages of high separation success rate, less time consumption, high purity, good proliferation activity, maintenance of differentiation potential in vitro long-term culture and the like. The ectopic endometrium mesenchymal stem cells obtained by the method can be used for basic research related to pathogenesis and application research of personalized targeted therapy, and new power is injected for advancing basic and clinical research of adenomyosis. Compared with papers in the background technology, the invention adopts a completely different strategy from the prior art, namely, firstly adopts a mode of low-speed 360 rotation of the complex enzyme digestive juice to digest and dissociate ectopic focus tissues for a short time (30 minutes), and the purpose of doing so is to digest extracellular matrixes and connective fibers on the surface layer of focus tissues, thereby helping to dredge a migration channel and facilitating the climbing out of intima cells in the subsequent tissue mass adherence culture. And simultaneously, the active damage to the endometrial mesenchymal stem cells with extremely low content in the tissue is reduced as much as possible. Then, the tissue adherence method is further used, the tissue subjected to primary digestion dissociation is firstly subjected to inverted adherence culture, and after the tissue blocks subjected to inverted culture are firmly adhered, the tissue blocks are subjected to normal culture until primary endometrial cells climb out and are converged. Finally, endometrial mesenchymal stem cells were isolated using PE-labeled SUSD2 antibodies according to conventional magnetic bead sorting.

Drawings

FIG. 1 is a flow chart of a technique for isolating and culturing eMSCs ectopic lesions of adenomyosis.

FIG. 2 is an in vitro culture observation of primary endometrial cells extracted from a ectopic focus of adenomyosis.

FIG. 3 detection of the positive rate of SUSD2+ eMSCs of primary endometrial cells extracted from a ectopic focus of adenomyosis;

(A) Analyzing SUSD2 expression of primary endometrial cells cultured by different separation methods by flow cytometry;

(B) Statistical analysis of SUSD2+ eMSCs occupancy in primary endometrial cells cultured by different isolation methods.

FIG. 4 detection of surface marker expression after subculturing of adenomyosis ectopic eMSCs.

FIG. 5 detection of the proliferation potency of adenomyosis ectopic eMSCs in vitro;

(A) CCK8 method detects eMSCs in vitro proliferation capability difference obtained by different separation methods;

(B) The EdU method detects eMSCs in vitro proliferation activity differences obtained by different separation methods.

FIG. 6 detection of ectopic eMSCs in vitro osteogenic differentiation potential of adenomyosis;

(A) The osteogenic differentiation kit detects eMSCs in vitro differentiation potential obtained by different separation methods;

(B) And (5) carrying out statistical analysis on total gray values of the calcium nodule staining positive areas after the osteogenesis induction is finished.

FIG. 7 shows the difference in the effects of eMSC s separation in tissue isolated from ectopic lesions of adenomyosis under different enzyme digestion systems;

(A) In vitro culture observation of primary endometrial cells separated under different enzyme digestion systems;

(B) Flow cytometry analysis of primary endometrial cell SUSD2 expression in different enzyme digestion systems;

(C) Statistical analysis of SUSD2+ eMSCs occupancy in primary endometrial cells cultured with different enzyme digestion systems.

Detailed Description

Example 1

1. Tissue specimen collection

The research scheme implemented by the patent of the invention has obtained the examination and approval of the institute of Chinese medicine and institute of China (attached to the university of Chinese medicine in Nanjing) and institute of China (batch number: 2023-LWKYZ-050). Patients were enrolled for selection of holatectomy for adenomyosis. The specific inclusion criteria are as follows: ① The uterine adenomyosis is identified by rapid pathological diagnosis after operation; ② The patient is not treated with hormones or other drugs prior to surgery; ③ The patients have no other reproductive endocrine diseases and malignant tumor diseases. After a total hysterectomy, the ectopic focal tissue in the myometrium is excavated under sterile conditions and placed in a 50 mL centrifuge tube containing precooled Phosphate Buffer (PBS), and immediately after the ice box is inserted, the tissue is transferred to a laboratory for primary endometrial cell isolation culture.

2. Primary endometrial cell isolation and culture

The specific operation steps are as follows

After the tissue specimen is transported to a laboratory, three different schemes are developed simultaneously for primary endometrial cell separation and culture, and the specific operation steps are as follows:

(1) Scheme one: enzymatic digestion

0.5 Cm ³ ectopic focal tissue (with obvious bleeding site position selected) was taken into a 5mL centrifuge tube and added with a little PBS, sheared to 1mm ³ size pieces with sterile scissors, then 3mL complex enzyme digest (consisting of 1.25 mg/mL trypsin and 0.5 mg/mL type IV collagenase) was added, placed in a low-speed rotary mixer (Shanghai damm Utility Co.) for digestion for 2 hours, and after stopping digestion 1000 r/min centrifugation for 3 minutes the supernatant was discarded, and the cell pellet was resuspended. 100. And (3) filtering a [ mu ] m cell sieve, collecting a cell suspension, centrifuging 3 min, and discarding the supernatant. Normal complete medium (consisting of 89% DMEM/F12, 10% foetal calf serum and 1% triple antibody solution) was added and resuspended in 10 cm ³ dishes. The dishes were placed in a 37℃,5% CO ₂ and saturated humidity incubator and the medium was changed every 2 days until the adherent endometrial cells grew to complete confluence.

(2) Scheme II: tissue block method

Taking 0.5 cm ³ ectopic focus tissue (with obvious bleeding point position selected) to a5 mL centrifuge tube, adding a little fetal bovine serum, aseptically shearing to 1 mm ³ size fragments, inoculating to a 10 cm ³ culture dish, and placing in an incubator for reverse buckling culture for 2 hours. The dishes were placed on, 5 mL normal complete medium was added, 24 h followed by normal complete medium to 10mL, 48 hours later PBS washing to remove non-adherent tissue pieces and 10mL complete medium was added. The dishes were placed in a 37 ℃,5% CO2 and saturated humidity incubator and the medium was changed every 2 days until endometrial cells in the tissue mass climbed out and grew to complete confluence.

(3) Scheme III: integrated process

0.5 Cm ³ ectopic focal tissue (with obvious bleeding point location selected) was taken into a 5mL centrifuge tube and added with a little PBS and aseptically sheared to 1 mm ³ size pieces. Then adding complex enzyme digestion solution (composed of 1.25 mg/mL trypsin and 0.5 mg/mL type IV collagenase), and placing on a rotary mixer arranged in a constant temperature incubator to digest 30min in a 10 rpm and 360 degree rotation mode. After digestion, the supernatant was removed by adding complete common medium to terminate digestion and centrifugation. The tissue pellet was resuspended in fetal bovine serum in a centrifuge tube and the suspension was spread in 10 cm cell culture dishes using a sterile pipette tip. Transferring the culture dish into an incubator, standing for 3 minutes, reversely buckling and culturing for 2 hours to ensure that the tissue blocks cultivated reversely are firmly adhered, turning the cell culture dish, placing the cell culture dish on the culture dish, adding 4 mL ordinary complete culture medium, and supplementing the ordinary complete culture medium to 10 mL after 24 hours. After 48 hours, the non-adherent tissue pieces were removed by PBS washing and 10 mL complete medium was added. The dishes were placed in 37℃and 5% CO ₂ and saturated humidity incubator and the medium was changed every 2 days until endometrial cells in the tissue mass climbed out and grew to complete confluence (see FIG. 1).

3. Comparison of separation effects of different separation schemes eMSCs

Observing the growth condition of the primary endometrial cells under different separation schemes under an inverted microscope, and simultaneously, utilizing an image acquisition system to carry out photographing record and analyzing the time required by the attachment and confluence of the primary endometrial cells; the flow cytometer was used to examine the eMSCs positive rate of primary endometrial cells grown to confluence under different isolation protocols and to analyze the effect of the different isolation protocols on the eMSCs positive rate (enrichment level) of primary endometrial cells grown to confluence.

3.1 Primary endometrial cell growth obtained from different primary endometrial cell isolation protocols

On days 1, 3, 5, 7 and 9 after the end of the tissue treatment described in the above 2, the primary endometrial cell growth obtained by the different isolation protocols was observed under an inverted microscope and recorded by photographing (see FIG. 2).

(1) First Day (Day 1)

Enzymatic digestion: the cell adhesion of the tadpole shape with the short round shape can be seen, and the cell extensibility of the adhered cell is general;

Tissue block method: larger tissue masses can be seen attached to the bottom of the dish, but no cell climbing out is seen;

the combination method comprises the following steps: the naked eye can see that a significant number of interstitial-like cells crawl out around the smaller tissue mass.

(2) Third Day (Day 3)

Enzymatic digestion: small numbers of scattered long spindle cells and clustered epithelial-like cells can be seen;

tissue block method: the large tissue block is adhered to the bottom of the dish, and no cell is seen to climb out;

The combination method comprises the following steps: the tissue mass becomes smaller and there begins to be a cluster of growing mesenchymal-like cells around.

(3) Fifth Day (Day 5)

Enzymatic digestion: the number of the mesenchymal cells with obvious quantity is obviously reduced, and the epithelial cells are obviously reduced;

Tissue block method: the larger tissue block is attached to the bottom of the dish, and interstitial-like cells start to climb out of the tissue;

the combination method comprises the following steps: there is more mesenchymal cells growing around the tiny tissue mass in a dispersive way.

(4) Seventh Day (Day 7)

Enzymatic digestion: the number of the mesenchymal cells is obviously increased before, and almost no epithelial-like cells are seen;

Tissue block method: more scattered mesenchymal cells were visible around the tissue mass and slightly further;

The combination method comprises the following steps: a large number of mesenchymal cells spirally grow around the tiny tissue mass.

(5) Ninth Day (Day 9)

Enzymatic digestion: the number of the mesenchymal cells is increased continuously before;

Tissue block method: a large number of interstitial-like cells can be seen around the tissue mass;

the combination method comprises the following steps: the cell growth situation is good (the cell grows in a vortex shape), and the cells growing at the dish bottom are nearly completely converged.

Subsequently, we performed statistics on the results of primary endometrial cell isolation from 10 cases of adenomyosis ectopic focal tissue, and the results showed that: (1) The success rate of separating primary endometrium cells by adopting a combination method (10/10, 100%) is significantly higher than that of a single enzyme digestion method (3/10, 30%) (P < 0.001) and a tissue mass method (4/10, 40%) (P < 0.01); (2) The primary endometrial cell attachment time (1.00.+ -. 0.00 days) obtained with the combination method is comparable to the enzymatic digestion method (1.33.+ -. 0.58 days) (P > 0.05), but significantly shorter than the tissue mass method (3.67.+ -. 1.15 days) (P < 0.01) (see Table 1); (3) Primary endometrial cell confluence times (6.67±0.58 days) obtained using the combination method were significantly shorter than the enzymatic digestion method alone (18.00±1.00 days) (P < 0.001) and the tissue mass method alone (22.67±0.58 days) (P < 0.001) (see table 2). In addition, the combined method showed a better morphology of primary endometrial cell growth than the enzyme digestion alone or the tissue mass in vitro isolation from Day 5, day 7 and Day 9 (see fig. 2).

TABLE 1 time to primary endometrial cell attachment

TABLE 2 Primary endometrial cell confluence time

The above results indicate that: the combined method can obtain the primary endometrial cells from the ectopic focus more quickly than the single enzyme digestion or tissue block method, and the obtained primary endometrial cells have better growth morphology.

3.2 Comparison of SUSD2+ eMSCs Positive Rate in primary endometrial cells under different isolation protocols

Still further, the surface molecular marker SUSD2 expression specifically expressed by eMSCs in the pooled primary endometrial cells of 5.1 (see a in fig. 3) was analyzed using flow cytometry and the statistical results were as follows: the SUSD2+ eMSCs positive rate (55.7+ -1.7%) obtained by the combined method when primary endometrial cells were grown to confluence was significantly higher than that obtained by the enzyme digestion method alone (18.0+ -1.1%) (P < 0.01) or tissue mass method (13.9+ -1.7%) (P < 0.01) (see B in FIG. 3 and Table 3);

TABLE 3 SUSD2+ eMSCs Positive Rate in primary endometrial cells

The above results indicate that: compared with the single enzyme digestion or tissue block method, the combined method has higher positive rate of eMSC s in primary endometrial cells, namely the combined method can obviously improve the number (enrichment level) of eMSCs absolute cells in primary endometrial cells before immunomagnetic bead separation, thereby greatly improving the efficiency of eMSCs in vitro separation, culture and amplification in adenomyosis ectopic focus.

Example 2 immunomagnetic bead method sorting eMSCs and amplification culture

When the primary endometrial cells described in example 1,2, were grown to complete confluence, eMSCs of the primary endometrial cells were isolated and expanded by immunomagnetic bead method. The specific operation steps are as follows:

1. Preparation of cell suspensions

(1) Taking out the primary endometrium cells after confluence, discarding the culture medium, and washing for 2 times by PBS;

(2) 0.25% Trypsin (Biosharp, BL 512A) digested for 10 min;

(3) Re-suspending by using an endometrial mesenchymal cell culture reagent, repeatedly blowing, and transferring into a 15 mL centrifuge tube;

(4) Centrifuging at 300 g for 3 min, and discarding supernatant;

(5) Add the appropriate amount of MACS Buffer (Miltenyi Biotec, 130-091-221) to resuspend and count cells;

(6) 300 g was centrifuged for 10min and the supernatant discarded.

2. Cell magnetic markers

(1) MACS Buffer was added at 95. Mu.L/10 ⁶ cells and resuspended;

(2) PE anti-human SUSD2 anti-ibody (BioLegend, 327406) was added at 5. Mu.L/10 ⁶ cells;

(3) Light shielding and incubation for 15 minutes at 4 ℃ in a refrigerator;

(4) Adding MACS Buffer to 4 mL, and washing;

(5) Centrifuging for 10min at 300 g, and discarding supernatant;

(6) MACS Buffer was added to the cells at 90. Mu.L/10 ⁶ for resuspension;

(7) Anti-PE microblads (Miltenyi Biotec, 130-048-801) was added at 10. Mu.L/10 ⁶ cells;

(8) The cell suspension is placed in a refrigerator at 4 ℃ and incubated for 15 minutes in a dark place;

(9) Washing with 1 mL MACS Buffer a;

(10) Centrifuging for 10 min at 300 g, and discarding supernatant;

(11) 1 mL MACS Buffer.

3. Immunomagnetic bead sorting

(1) MS separation columns (Miltenyi Biotec, 130-122-727) were mounted on MACS separators;

(2) Slowly adding 500 mu L of MACS Buffer into the sorting column for rinsing for 1 time to avoid generating bubbles;

(3) The liquid in the separation column is drained, and the cell suspension is added into the separation column for times according to 500 mu L/time;

(4) Cells not bound to the magnetic beads were washed by adding MACS Buffer (3 times) to the column at 500. Mu.L/time.

(5) Taking down the MS sorting column and placing the MS sorting column on a new 15 mL centrifuge tube;

(6) Adding 1 mLMACS Buffer into the sorting column, uniformly pressing down the piston with force, and collecting cell suspension flowing out from the lower end of the sorting column;

(7) 300 g centrifugal 5 minutes of the above cell suspension, discarding the supernatant to retain cell sediment, then PBS washing 1 time, discarding the supernatant to retain cell sediment.

(8) EMSCs complete medium consisting of 10% FBS (Viva cell, C04400-500), 1 XPen-Strep-Ampho.B (Gibco, 15240-062), 2mM GlutaMax ^TM and DMEM/F12 was prepared.

(9) Cell pellet was resuspended in eMSCs complete medium and plated onto cell culture dishes for expansion culture.

(10) When eMSCs is full, the method comprises the following steps of 1:3 continuing to use the stem cell culture reagent for amplification culture.

EXAMPLE 3 evaluation of eMSCs surface molecular marker expression and in vitro proliferation Activity and differentiation potential obtained by different isolation protocols

Further, when eMSCs obtained in example 2 was expanded in vitro to passage 6 (P6), fraction eMSCs was used to analyze the effect of different isolation protocols on the expression, proliferation activity and osteogenic differentiation potential of the in vitro expanded cultured eMSCs surface molecular markers (CD 73, CD90 and CD 105).

1 EMSCs surface molecular marker expression

Flow cytometry detection: the cells were resuspended in log phase growth eMSCs (passage 6 in each case), pancreatin digested, centrifuged, and the concentration was adjusted to 1X 10 ⁷ cells/mL using pre-chilled PBS (3% BSA). mu.L of cell suspension (1X 10 ⁶) was taken, and mouse anti-human CD73-PE, CD90-PE, CD105-PE, CD45-PE, HLA-DR-PE and the corresponding isotype control antibodies were added respectively and thoroughly mixed. Incubate at 4℃for 15 min in the dark, centrifuge 5 min g and discard the staining solution to leave a pellet. The cells were resuspended in 500 μl PBS, immediately detected by BD FACSVerse flow cytometry, and analyzed using FlowJo (Version 10) software after the raw data were derived.

The surface positive molecular markers (CD 73, CD90 and CD 105) and negative control molecules (CD 45 and HLA-DR) were expressed using flow cytometry analysis of eMSCs (all passage 1. Note: eMSCs flasks obtained by magnetic bead sorting and cells collected after first digestion, centrifugation after confluence were noted passage 1) obtained from different primary endometrial cell isolation protocols (see FIG. 4), and the results showed that: eMSCs surface molecules CD73, CD90 and CD105 obtained by different separation schemes are all strong positive, and have no obvious difference in expression level (average P > 0.05) (see Table 4); furthermore, the eMSCs surface molecules finally obtained from the different primary endometrial cell isolation protocols were nearly negative for CD45, HLA-DR expression (see Table 4).

TABLE 4 eMSCs expression of surface molecular markers

The above results indicate that: the expression of eMSCs surface stem cell molecular markers obtained by different primary endometrial cell separation schemes is not obviously different, namely, the separation scheme by a combination method is the same as the traditional single enzyme digestion or tissue block method, and the expression of eMSCs surface molecular markers obtained after magnetic bead separation is not influenced.

2 EMSCs in vitro proliferation Activity

(1) CCK-8 experiment: CCK-8 is known as Cell Counting Kit-8 reagent, and can be used for simple and accurate cell proliferation and toxicity analysis. The basic principle is as follows: the reagent contains water-soluble tetrazolium salt WST-8[ chemical name: 2- (2-Methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazolium monosodium salt ], which is reduced by dehydrogenase in the cell to a yellow formazan product (Formazan dye) with high water solubility under the action of the electron carrier dimethyl 1-Methoxy-5-methylphenazinium sulfate (1-Methoxy PMS). The amount of formazan produced was proportional to the number of living cells. Thus, this property can be used to directly conduct cell proliferation and toxicity analysis.

The cells were grown eMSCs (all 6 th generation) in log phase, trypsinized, centrifuged, resuspended in eMSCs complete medium and seeded into 96-well plates at a density of 2×10 ⁵/mL, 3 wells per group, 10 μl CCK8 solution (Biosharp) was added to each well of cells at culture numbers 0, 24, 48, 72 and 96 h, and absorbance (OD value) at 450nm wavelength was measured with an enzyme-labeled instrument after incubation for 2 h. The above experimental procedure was repeated 3 times and the average was plotted.

(2) EdU experiment: edU (5-Ethynyl-2' -deoxyuridine) is a thymidine analog capable of substituting thymine for being incorporated into replicating DNA molecules during the cell proliferation phase, and the proliferation capacity of cells can be rapidly and accurately detected by rapidly detecting the replication activity of cellular DNA based on the specific reaction of EdU with Apollo ^® fluorescent dye.

The logarithmic phase was taken for growth eMSCs (both 3 rd generation), pancreatin digested, centrifuged, resuspended in eMSCs complete medium and inoculated into 96-well plates at a density of 5×10 ⁵/mL and cultured to the normal growth phase. The whole culture medium is used according to the proportion of 1000:1 (RiboBio) to prepare a proper amount of 50 mu M EdU culture medium, adding 100 mu L of EdU culture medium into each hole to incubate for 2 hours, washing with PBS for 1-2 times, and each time for 5 min; the 4% paraformaldehyde is fixed at room temperature for 30min, 50 mu L of 2 mg/mL glycine is added to each well, the mixture is incubated for 5 minutes by a decolorizing shaker, 100 mu L of PBS is added to each well, the mixture is washed for 5 minutes by the decolorizing shaker, 100 mu L of 0.5% Triton X-100 PBS is added to each well, the mixture is incubated for 10min by the decolorizing shaker, and the mixture is washed for 5 minutes by the PBS. Adding 100 mu L of 1X Apollo dyeing reaction liquid into each hole, incubating for 30min by a light-proof room temperature decoloration shaker, adding 100 mu L of 0.5% Triton X-100 PBS decoloration shaker, and cleaning for 2-3 times, each time for 10 minutes; 100 mu L of 1 Xhoechst 33342 reaction solution is added into each hole, the mixture is incubated for 30 minutes by a light-proof, room temperature and decoloration shaking table, 100 mu L of PBS is added into each hole for cleaning 1-3 times, and the mixture is immediately photographed under a fluorescence microscope after the completion of dyeing.

Evaluation of in vitro cell proliferation activity was performed on eMSCs (both passage 6) obtained from different primary endometrial cell isolation protocols using CCK-8 and EdU methods, and the results showed that: (1) CCK-8 detection results show that eMSCs obtained by the combined method has significantly stronger in vitro proliferation capacity than that obtained by the enzyme digestion method or the tissue mass method alone (see A in FIG. 5); (2) The EdU experimental results show that eMSCs obtained by the combined method has significantly stronger proliferation activity in vitro than that obtained by the enzyme digestion method and the tissue mass method alone (see B in FIG. 5). The results show that: the eMSCs in vitro proliferation activity obtained by the combined method is stronger than that obtained by the single enzyme digestion method or the tissue block method.

3 EMSCs in vitro osteogenic differentiation potential

EMSCs was subjected to osteoinductive differentiation according to the instructions of the human-related stem cell osteoinductive differentiation kit (OriCell, HUXXC-90021). First, eMSCs (each generation 3) to be induced was inoculated into a six-well plate at a cell density of 2X 10 ⁴ cells/cm ², and cultured in a CO2 incubator at 37℃with 5% CO2 and saturated humidity. When the cell fusion reached 70%, 2 mL osteogenic differentiation medium was added to the six well plate. Fresh stem cell osteogenic differentiation medium was replaced every 3 days. And then, after induction for 2-4 weeks, observing cell morphology change and bone matrix calcification nodule formation, carrying out alizarin red staining on different induced cells at the same time point, observing the staining effect under a microscope, and photographing. Finally, total gray values of calcium nodule positive staining areas were analyzed using Image J (Version 1.43 u) Image processing software (INTEGRATED DENISITY, intDen), statistically analyzed using GRAPH PAD PRISM (Version 9.0) software and plotted.

Evaluation of osteogenic differentiation potential was performed on eMSCs (both generation 6) obtained from different primary endometrial cell isolation protocols using a human-associated stem cell osteogenic differentiation kit, and the results showed that: the combined method achieved eMSCs in vitro osteoinduction with significantly higher gray scale values for calcium nodule staining than either the enzymatic digestion method alone or the tissue mass method alone (see a and B in fig. 6). The results show that: the eMSCs in vitro proliferation capacity obtained by adopting the combination method is stronger than that obtained by adopting the single enzyme digestion method or the tissue block method.

Example 4 differential effects of eMSC s separation in ectopic focus tissue of adenomyosis in different enzyme digests

1. Tissue specimen collection

The research scheme implemented by the patent of the invention has obtained the examination and approval of the institutional review board of the traditional Chinese medicine institute of Jiangsu province (combination of traditional Chinese and western medicine of Jiangsu province) (batch number: 2023-LWKYZ-050). Patients were enrolled for selection of holatectomy for adenomyosis. The specific inclusion criteria are as follows: ① The uterine adenomyosis is identified by rapid pathological diagnosis after operation; ② The patient is not treated with hormones or other drugs prior to surgery; ③ The patients have no other reproductive endocrine diseases and malignant tumor diseases.

Under the same condition that the patient is informed before operation, after the holectomy, ectopic focus tissues in the myometrium are excavated under aseptic conditions and put into a 50mL centrifuge tube (precooled in advance) containing Phosphate Buffer Solution (PBS), and immediately after the ice box is inserted, the ectopic focus tissues are transferred to a laboratory for primary endometrial cell separation culture.

2. Primary endometrial cell isolation and culture

After the tissue specimen is transported to a laboratory, the primary endometrial cells are separated and cultured by a simple enzyme digestion method, and the specific operation steps are as follows:

3 parts of 0.5 cm ³ ectopic focus tissues are respectively placed in 5mL centrifuge tubes, a little PBS is added, the focus tissues are sheared to be 1 mm ³ size by sterile scissors, then 3 mL different tissue digests (digestive fluid 1:1.25 mg/mL trypsin; digestive fluid 2:0.5 mg/mL type IV collagenase; digestive fluid 3: consisting of 1.25 mg/mL trypsin and 0.5 mg/mL type IV collagenase) are respectively added, the focus tissues are placed in a low-speed rotary mixer (Shanghai damm industry Co., ltd.) for digestion for 2 hours, and after digestion is terminated, the supernatant is discarded by centrifugation for 3 minutes at 1000 r/min, and cell precipitation is resuspended. 100. And (3) filtering a [ mu ] m cell sieve, collecting a cell suspension, centrifuging 3 min, and discarding the supernatant. The cell pellet was resuspended in normal complete medium (consisting of 89% DMEM/F12, 10% fetal bovine serum and 1% triple antibody solution) and plated onto 10 cm dishes. The dishes were placed in a 37℃,5% CO ₂ and saturated humidity incubator and the medium was changed every 2 days until the cells grew to complete confluence.

3. Analysis of results

From the viewpoint of the primary endometrium cell growth morphology, the primary endometrium cell growth state obtained by separation with digestive juice 3 is significantly better than that obtained by separation with digestive juice 1 and digestive juice 2 (see a in fig. 7).

The time required for primary endometrial cells obtained by separation using digestive juice 3 to grow to confluence (17.33.+ -. 1.53 days) was significantly shorter than the time required for primary endometrial cells obtained by separation using digestive juice 2 to grow to confluence (23.67.+ -. 2.08 days) (P < 0.05), whereas primary endometrial cells could not be successfully obtained using digestive juice 1, from the time required for primary endometrial cell paste separation to grow to complete confluence.

From the aspect of the success rate of primary endometrium cell separation, the success rate of primary endometrium cell separation by using the digestive juice 3 is higher than that of primary endometrium cell separation by using the digestive juice 2 (2/10, 20%), and the primary endometrium cell separation by using the pancreatic protein digestion method alone cannot be successfully obtained finally, namely, the success rate of primary endometrium cell separation by using the pancreatic protein digestion method (0/10, 0%).

Analysis of the eMSCs positive rate of primary endometrial cells obtained by the above-described differential separation of digestive juice using flow cytometry, when grown to confluence, showed that the susd2+ eMSCs positive rate (18.47±1.27%) was significantly higher than that of digestive juice 2 (13.00±1.02%) after confluence of primary endometrial cells obtained with digestive juice 3 (see B in fig. 7).

The above results indicate that: the enzyme digestion system (digestive juice 3) adopting the combination of trypsin and collagenase has significantly better separation effect for eMSCs in the ectopic focus tissue of the adenomyosis than the enzyme digestion system adopting trypsin or collagenase alone.

Summary of example effects

The effects exhibited by examples 1-4 above can be found: the method can be used for efficiently extracting the endometriosis focus endometrium mesenchymal stem cells, and the obtained endometrium mesenchymal stem cells can still maintain the form, the surface molecular marker expression, the in-vitro proliferation activity and the directional differentiation potential of the mesenchymal stem cells after in-vitro passage expansion for a plurality of times. Compared with the prior art, the extraction method has the most remarkable advantages that: the method has the advantages of short cell extraction period, high overall separation success rate and simple operation technology, can realize rapid acquisition of a large number of ectopic endometrial mesenchymal stem cells, and lays a solid foundation for developing basic research related to pathogenesis of adenomyosis and application research of personalized cell targeted therapy based on the ectopic endometrial mesenchymal stem cells.

Claims

1. A method for extracting endometrium mesenchymal stem cells in endometriosis ectopic focus, which is characterized in that the method adopts a method of combining an enzyme digestion method with a tissue block method to extract primary endometrium cells in adenomyosis ectopic focus, and then uses PE marked SUSD2 antibody to separate the endometrium mesenchymal stem cells according to a conventional magnetic bead separation method, wherein the enzyme digestion method is as follows: adding the complex enzyme digestion solution into a container for holding tissues, and placing the container on a rotary mixer arranged in a constant temperature incubator for digestion and dissociation; the tissue block method is as follows: centrifuging after the enzyme digestion method is finished, discarding the supernatant, reserving tissue sediment, adding a small amount of serum to resuspend the tissue, dispersing and coating the tissue in a culture dish by using a sterile suction head, transferring the culture dish into an incubator to start inverted culture, overturning the cell culture dish after the tissue blocks subjected to inverted culture are firmly attached, and simultaneously adding a complete culture medium to perform normal culture until intima cells around the tissue blocks climb out and grow to complete confluence; finally, endometrial mesenchymal stem cells were isolated using PE-labeled SUSD2 antibodies according to conventional magnetic bead sorting.

2. The method for extracting endometrium mesenchymal stem cells from ectopic focus of adenomyosis according to claim 1, wherein the complex enzyme digestive juice consists of trypsin with a final concentration of 1-1.5 mg/mL and collagenase type IV of 0.25-0.75 mg/mL.

3. The method for extracting endometrium mesenchymal stem cells from ectopic focus of adenomyosis according to claim 2, wherein the special complex enzyme digestive juice consists of trypsin with final concentration of 1.25 mg/mL and collagenase type IV of 0.5 mg/mL.

4. A method of extracting endometrial mesenchymal stem cells from ectopic lesions of adenomyosis according to claim 1, wherein the complex enzyme digest time is controlled between 15 and 45 minutes, preferably 30 minutes.

5. The method for extracting endometrial mesenchymal stem cells from ectopic foci of adenomyosis according to claim 1, wherein the container is placed on a rotary mixer arranged in a constant temperature incubator in an enzyme digestion method, and low-speed 360-rotation digestion dissociation is adopted; the low speed is a rotation speed lower than 20 rpm; preferably a rotational speed of 10 rpm.

6. A method for extracting endometrial mesenchymal stem cells from ectopic lesions of adenomyosis according to claim 1, wherein the tissue mass procedure is performed within 15 minutes after the end of the enzymatic digestion procedure, preferably immediately after the end of the enzymatic digestion procedure.

7. A method of extracting endometrial mesenchymal stem cells from ectopic foci of adenomyosis according to claim 1, wherein the small amount of added serum is used to resuspend the tissue and spread evenly in 10 cm dishes using a sterile suction head, and the rest time of subsequent transfer to the incubator is controlled between 1 and 5 minutes, preferably 3 minutes.

8. A method of extracting endometrial mesenchymal stem cells from ectopic foci of adenomyosis according to claim 1, wherein transferring the petri dish to an incubator starts an inversion culture. Wherein the inversion culture time is controlled between 1 and 3 hours, preferably the inversion culture time is 2 hours.

9. The method for extracting mesenchymal stem cells from ectopic foci of adenomyosis according to claim 1, wherein when the tissue mass is firmly adhered, the tissue mass is inverted and then the cell culture dish is turned over for normal culture by 10 cm, and the complete culture medium is added until the endometrium cells around the tissue mass climb out and grow to complete confluence, and the volume of the complete culture medium is 2 to 6 mL.

10. Use of any one of the methods of extracting endometrial mesenchymal stem cells in the ectopic focus of adenomyosis according to claims 1-9 in basic research and targeted therapy.