KR102312361B1 - Catheter for collecting endometrial tissue, and method for obtaining endometrium-derved stem cell using thereof - Google Patents

Abstract

The present invention relates to a catheter for collecting endometrial tissue and a method for obtaining stem cells from endometrial tissue using the same. It is provided with an insertion unit 100 having at least one 101, and connected to the other open end of the insertion unit 100, and has an outlet 201 for discharging the introduced endometrial tissue and menstrual blood. Collecting endometrial tissue using a catheter including a storage and discharge unit 200 and a stopper 210 for opening and closing the outlet 201, and crushing the collected endometrial tissue And, ultrasonically treating the crushed endometrial tissue, and centrifuging the ultrasonically treated endometrial tissue to separate cells. The catheter for collecting endometrial tissue according to the present invention and the method for obtaining stem cells from endometrial tissue using the same can easily collect endometrial tissue without damage, and can be stored for a long time because a preservative solution can be added immediately. , there is the convenience of transport, there is no damage to the cells, and the survival rate is excellent, so the quality of the mesenchymal stem cells can be improved. In addition, there is an advantage in that the process time can be shortened and the efficiency can be increased.

Description

Catheter for collecting endometrial tissue and method for obtaining stem cells from endometrial tissue using the same

The present invention relates to a catheter for collecting endometrial tissue and a method for obtaining stem cells from endometrial tissue using the same.

Stem cell refers to a cell that has the ability to differentiate into two or more cells while having the ability to self-replicate. In general, stem cells include embryonic stem cells (ESCs) and adult stem cells, depending on the origin of the cells. It can be classified into cells (Adult Stem Cells, ASCs).

Adult stem cells tend to be replaced by the term Somatic Stem Cells (SSCs) as it has been confirmed that stem cells isolated from fetal tissues have different characteristics from stem cells isolated from true adults. However, stem cells isolated from fetuses or adults all meet the criteria for defining mesenchymal stem cells (MSCs).

Endometrial tissue-derived stem cells are a type of MSCs derived from endometrial tissue. MSCs are also called mesenchymal stem cells or adult stem cells, and collectively refer to stem cells isolated and cultured from tissues other than embryos. They exist in small amounts in most tissues that have already been differentiated, and their presence has been confirmed in almost all tissues studied so far, such as umbilical cord blood, umbilical cord, teeth, eyes, placenta, hair follicles, lungs, and liver. As described above, mesenchymal stem cells are present in almost all parts of the body, but it is very difficult to distinguish them as tissue-specific markers or expressed genes. According to the definition, mesenchymal stem cells, when isolated and cultured, grow attached to a culture dish, express CD105, CD73, and CD90, do not express CD45, CD34, CD14, CD79, and HLA-DR, and are mesoderm tissue ( bone, cartilage, fat) (Fukuchi Y et al, Stem Cells 22:649-658, 2004; Wulf GG et al, Tissue Eng 10:1136-1147, 2004; Dominnici M et al, Cytotherapy 8 :315~317, 2006) However, according to recent studies, it has been found that endometrial stem cells can differentiate not only into mesoderm but also into ectoderm (mainly nerve cells) or endoderm (hepatocytes, pancreatic cells, etc.). It is recognized to have pluripotency (Multipotency).

In a conventional method for obtaining endometrial tissue-derived stem cells, the endometrial tissue extracted during endometriosis is washed, treated with a collagenase solution at 37° C. for 60 minutes, and centrifuged to obtain stem cells. is to obtain

In addition, a menstrual cup is mounted on a woman's vagina for 2-3 hours, the endometrial tissue and menstrual blood received in the menstrual cup are collected, and the endometrial tissue is separated therefrom, followed by a collagenase solution. Processed at 37° C. for 60 minutes, and centrifuged to obtain stem cells.

However, this conventional method has disadvantages in that it takes a long time, and a research reagent called collagenase is used to damage cells, and the survival rate is lowered.

In addition, the method of collecting endometrial tissue using a menstrual cup requires at least 3 hours or more to collect the tissue, requires two or more procedures such as inserting and removing the menstrual cup, and there is a high possibility of contamination of the endometrial tissue. , there was a problem with a high probability of tissue damage.

Therefore, there is a need for a method for collecting endometrial tissue by a simple method and obtaining mesenchymal stem cells from the endometrial tissue without damaging the cells.

KR 10-2018-0012223 A KR 10-1533789 B1

Fukuchi Y et al, Stem Cells 22:649-658, 2004 Wulf GG et al, Tissue Eng 10:1136~1147, 2004 Dominnici M et al, Cytotherapy 8:315-317, 2006 Science 276, 71-74, 1997; Science 284, 143-147, 1999; Science 287, 1442-1446, 2000 Proc Natl Acad Sci USA 96, 14482-14486,1999; Nature 401, 390-394, 1999 Tissue Eng 7, 211-228, 2001

Accordingly, an object of the present invention is to provide a catheter for collecting endometrial tissue that can easily collect endometrial tissue and menstrual blood without contamination and damage.

Another object of the present invention is to provide a catheter for endometrial tissue collection that can collect and separate endometrial tissue and menstrual blood at a time, and facilitate storage and movement of the collected endometrial tissue and menstrual blood.

Another object of the present invention is that the endometrial tissue can be isolated from the endometrial tissue only by ultrasonication without the use of collagenase, thereby reducing cell damage, increasing the survival rate, and shortening the process time. An object of the present invention is to provide a method for obtaining stem cells from a tissue.

The catheter for collecting endometrial tissue according to the present invention for achieving the above object has a tubular shape, and at least one inlet through which the endometrial tissue and menstrual blood can flow into the rear of the open end is provided with an insertion unit provided with at least one catheter and a storage and discharge unit provided at the other open end of the insertion unit and provided with an outlet for discharging the introduced endometrial tissue and menstrual blood, and a stopper for opening and closing the outlet, wherein the insertion unit passes through the cervix It is characterized in that it is inserted into the uterus.

The storage and discharge unit is in the form of a balloon having an extension formed to surround a part of the other side of the insertion unit, and the storage and discharge unit in the form of a balloon is provided with a separation means connected to the other open end of the insertion unit therein, the separation means It is characterized in that a plurality of through-holes are formed to separate the endometrial tissue from the endometrial tissue and menstrual blood introduced through the insertion part.

On the outside of the storage and discharge unit in the form of a balloon, it is characterized in that the removal string for the removal of the catheter is further provided.

And the method for obtaining stem cells from endometrial tissue according to the present invention comprises the steps of collecting endometrial tissue, chopping the collected endometrial tissue, and ultrasonically treating the crushed endometrial tissue. and centrifuging the sonicated endometrial tissue to isolate cells.

The ultrasonic treatment is characterized in that it is performed for 5 to 10 minutes at 10 to 100 kHz.

The catheter for collecting endometrial tissue according to the present invention and the method for obtaining stem cells from endometrial tissue using the same can easily collect endometrial tissue without damage, and can be stored for a long time because a preservative solution can be added immediately. , there is the convenience of transport, there is no damage to the cells, and the survival rate is excellent, so the quality of the mesenchymal stem cells can be improved. In addition, there is an advantage in that the process time can be shortened and the efficiency can be increased.

1 is a perspective view of a catheter according to a first embodiment of the present invention;
Figure 2 is a view showing the use state of Figure 1;
3 is a perspective view of a catheter according to a second embodiment of the present invention.
Figure 4 is a view showing the use state of Figure 3;
5 is a perspective view showing a third embodiment of the present invention.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

It is revealed that the stem cells in the present invention are mesenchymal stem cells derived from endometrial tissue.

First, the catheter for collecting endometrial tissue according to the present invention will be described.

The catheter for collecting endometrial tissue according to the present invention collects endometrial tissue extracted during endometriosis, or collects endometrial tissue and menstrual blood received after wearing a menstrual cup in a woman's vagina for 2 to 3 hours in the prior art. This is to solve problems such as contamination, tissue damage, and troublesome procedures during collection, and the biggest feature is that endometrial tissue can be easily collected without contamination and damage by using a catheter.

In addition, it is characterized in that it is possible to add a preservative solution immediately, so that it can be stored for a long time and there is convenience in transport.

1 and 2 are views according to a first embodiment of a catheter according to the present invention, as in FIGS. 1 and 2 , the catheter has a tubular shape, and endometrial tissue and menstrual blood flow into the rear of the open end. The insertion part 100 is provided with at least one inlet 101 through which it can be formed, and the outlet 201 is provided at the other open end of the insertion part 100, and discharges the introduced endometrial tissue and menstrual blood. ) includes a storage and discharge unit 200 formed therein, and a stopper 210 for opening and closing the outlet 201 , wherein the insertion unit 100 is inserted into the uterus through the cervix of a woman.

First, the insertion unit 100 is inserted into the uterus through a woman's vagina and cervix to obtain endometrial tissue and menstrual blood discharged from the uterus during menstruation. At this time, the insertion part 100 may have a tubular shape, and endometrial tissue and menstrual blood may be obtained through an open end, that is, the tip, and endometrial tissue and Menstrual blood can also be obtained.

In the present invention, the insertion part 100 is not limited in its length or internal and external diameter, but it is natural that it should have a length and diameter sufficient to obtain endometrial tissue and menstrual blood in the uterus. Illustratively, it is sufficient that the insertion part 100 has a length of about 25 cm, an inner diameter of 1.5 mm, and an outer diameter of about 2.5 mm.

In addition, since the insertion part 100 is a part to be inserted into the uterus, it is sufficient as long as the material is a transparent silicone material that is harmless to the human body and has some hardness, but is not necessarily limited thereto.

The storage and discharge unit 200 provided at the other open end of the insertion unit 100 is provided to communicate with the inside of the insertion unit 100 , and the endometrial tissue introduced through the insertion unit 100 . It stores and discharges menstrual blood. In the present invention, the size, shape, and material of the storage and discharge unit 200 are not limited, and may be formed integrally with the insertion unit 100 or may be separately formed and connected. However, on one side of the storage and discharge unit 200, a discharge port 201 for discharging endometrial tissue and menstrual blood is formed in a direction opposite to the direction in which the insertion unit 100 is provided. The size of is preferably a size corresponding to the inlet of a general syringe. This is because it is easy to use to discharge the endometrial tissue or menstrual blood of the storage and discharge unit 200 through a syringe.

And a stopper 210 for opening and closing the outlet 201 is additionally provided. Since the stopper 210 only needs to be able to open and close the outlet 201, the material, shape, etc. are not limited, and the stopper connection part 211 formed on the outer surface of one side of the storage and discharge unit 200 for convenience of use. It may be a form connected through, but is not limited thereto.

The catheter according to the first embodiment configured as described above is used as follows.

First, a speculum is inserted into the vagina of a woman who is menstruating to widen the vagina, and the vagina and cervix are disinfected. And with the stopper 210 of the catheter closed, the insertion part 100 is pushed into the uterus through the cervix. And when the stopper 210 of the catheter is opened and a separate syringe 1 is inserted into the outlet 201 of the storage and discharge unit 200 to create a negative pressure, as shown in FIG. 2 , the endometrial tissue in the uterus by the negative pressure The menstrual blood flows into the insertion unit 100 through the inlet 101, passes through the storage and discharge unit 200, and flows into the syringe 1 connected to the outlet 201, whereby endometrial tissue and menstrual blood are collected. this will be completed And the menstrual blood is separated from the endometrial tissue through a separate filter paper.

When endometrial tissue is collected through the above process, there is an advantage in that the convenience of collection is excellent and there is little damage to tissues and cells.

Figure 3 is a perspective view of a catheter according to a second embodiment of the present invention, Figure 4 is a view showing a state of use according to the second embodiment of the present invention, the configuration of the insertion part 100, stopper 210, etc. Since it is the same as that of the first embodiment, an additional description thereof will be omitted.

The second embodiment according to the present invention is characterized in that the storage and discharge unit 200 is in the form of a balloon, so it has the advantage of being able to create a negative pressure by itself without using a separate syringe 1 . At this time, as long as the balloon-shaped storage and discharge unit 200 is made of a material capable of providing negative pressure in the uterus by applying an external force to remove air in the balloon, it is most preferable to use, for example, a silicone material.

In addition, it is preferable that the storage and discharge unit 200 in the form of a balloon includes a separation means 220 connected to the other open end of the insertion unit 100 therein. It is separated from menstrual blood.

Specifically, the separation means 220 is for separating the obtained endometrial tissue and menstrual blood into endometrial tissue and menstrual blood, and has a form in which a plurality of through-holes 221 with a size of 0.5 mm are formed.

That is, only menstrual blood among the endometrial tissue and menstrual blood introduced through the insertion unit 100 passes through the through hole 221 and flows into the storage and discharge unit 200 , and the endometrial tissue is separated by the separation means 220 . ), so the separation of menstrual blood is completed only by the collection operation. In addition, since the separation means 220 also needs to create a negative pressure together with the storage and discharge unit 200, a material capable of providing negative pressure in the uterus by applying an external force to remove air in the balloon, for example, a silicone material is used. Most preferred.

However, in the second embodiment, the storage and discharge unit 200 is formed to extend by a predetermined length toward the open end of the insertion unit 100 , that is, an extension formed to surround a part of the other side of the insertion unit 100 . It may be in the form of a balloon provided with 230 , which is for ease of manufacture. Illustratively, the insertion unit 100 and the separation means 220 are integrally formed, and the storage and discharge unit 200 is formed separately, and the storage and discharge unit 200 is formed through the discharge port 201 inside the storage and discharge unit 200 . It is manufactured by inserting the inserting part 100 and the separating means 220 into the . At this time, it is preferable that the inner surface of the extension part 230 and the outer surface of the insertion part 100 are spaced apart from each other by a predetermined distance for easy discharge of the obtained menstrual blood. In addition, for easy discharge of the obtained endometrial tissue, it is preferable that the separation means 220 also have an outlet 222 at a position corresponding to the outlet 201 .

The catheter according to the second embodiment is used as follows.

First, as described above, the vagina of a woman who is menstruating is widened with a colposcopy, and then the vagina and cervix are disinfected. And after applying an external force to the balloon-shaped storage and discharging unit 200 and the separating means 220 of the catheter to create a negative pressure, with the stopper 210 closed, the insertion unit 100 of the catheter is pushed into the uterus put it in Then, the endometrial tissue and menstrual blood are introduced into the insertion unit 100 and the separating means 220 by the negative pressure, and the introduced endometrial tissue remains in the separating means 220, and the menstrual blood is stored and discharged in the separating unit 220. discharged into

When the collection process as described above is completed, the rear of the one end of the insertion unit 100, more specifically, the rear of the inlet 101, and the storage and discharge unit 200 is not covered by a separate After installing an opening/closing means (not shown) such as a clip to prevent the endometrial tissue from flowing back toward the insertion part 100 , the collected menstrual blood is transferred to the inner surface of the extension part 230 of the storage and discharge part 200 and the It is discharged through the spaced gap between the outer surfaces of the insertion part (100). Then, the stopper 210 of the storage and discharge unit 200 is opened and the obtained endometrial tissue is discharged through the outlets 201 and 222 .

In addition, when it is desired to store the collected endometrial tissue without using it immediately, the opening/closing means is installed in the extension part 230 of the storage and discharge unit 200, and is stored through the outlets 201 and 222. After injecting the solution, if the stopper 210 is closed, the endometrial tissue and menstrual blood can be stored for a long time without damage, thereby increasing the convenience of storage and preservation.

5 is a third embodiment according to the present invention. The catheter according to the third embodiment has the same shape and configuration as the second embodiment, but the length of the insertion part 100 is as short as about 7 cm. It is formed and inserted into the vagina until the balloon-shaped storage and discharge unit 200 .

The catheter according to this third embodiment inserts the insertion part 100 and the storage and discharge part 200 into the vagina while forming a negative pressure in the balloon in the same way as in the second embodiment, so that the insertion part 100 is inserted into the uterus. After being inserted, when endometrial tissue and menstrual blood are collected by negative pressure, the catheter is taken out from the inside of the vagina through the removal string 300 provided on the outside of the storage and discharge unit 200 .

The specific configuration of the catheter according to the third embodiment is the same as the second embodiment except for the length of the insertion part 100 and the strap 300 for removal, so a detailed description thereof will be omitted.

Hereinafter, a method for obtaining stem cells from endometrial tissue according to the present invention will be described in detail.

The method for obtaining stem cells according to the present invention includes the steps of collecting endometrial tissue, crushing the collected endometrial tissue, and ultrasonically treating the crushed endometrial tissue; It characterized in that it comprises the step of separating the cells by centrifuging the treated endometrial tissue.

Step of collecting endometrial tissue

First, endometrial tissue is collected using the catheter described above. Then, the collected endometrial tissue is washed with PBS (Phosphate Buffered Saline) or the like under aseptic conditions. At this time, when the endometrial tissue and menstrual blood are collected together, it is natural that menstrual blood can be separated from the endometrial tissue using filter paper or the like.

In this case, the collection of endometrial tissue may be done by other methods, but it is most preferable to use the catheter according to the present invention.

Crushing the collected endometrial tissue (chopping)

Next, the collected endometrial tissue is crushed (chopping). At this time, the size of the bar is not limited, it depends on the degree posted in the prior art.

Ultrasonic treatment of the crushed endometrial tissue

Then, the finely shredded endometrial tissue is treated with sonication to separate cells. That is, the conventional endometrial tissue isolates cells by using a collagenase solution, but the collagenase solution has disadvantages in that the treatment time is long and the cell viability is not high. The present invention is to isolate cells without damage within a short time by improving these disadvantages through sonication.

At this time, the ultrasonic treatment is preferably performed for 5 to 10 minutes at 10 to 100 kHz, because if the intensity of the ultrasonic treatment is out of this, the cells are damaged or the yield of stem cells is reduced.

In addition, the ultrasonic treatment is preferably performed in a state in which the endometrial tissue is suspended in the culture solution by adding a culture solution to the crushed endometrial tissue, and the amount of the culture solution added is such that the crushed endometrial tissue can be sufficiently suspended it is enough In this case, it is sufficient to use a conventionally published cell culture medium as the culture medium.

Separating the cells by centrifuging the sonicated endometrial tissue

Next, the sonicated endometrial tissue is centrifuged to separate cells. In this case, the centrifugation method is sufficient according to the method disclosed in the prior art.

And by culturing the isolated cells to obtain a stem cell, the culture method is according to the conventionally published method.

The method for obtaining endometrial tissue-derived stem cells by the above method has advantages in that the process time is shortened because a collagenase solution is not used, and the cell viability is high.

Hereinafter, the present invention will be described in detail through specific examples.

(Example 1)

Endometrial tissue was collected with a catheter according to the present invention. Then, it was washed with PBS (Phosphate Buffered Saline) under aseptic conditions, cut into pieces of 10 mm 3 or less, and then 10% Fetal Bovine Serum (FBS), 0.1 mM beta-mercaptoethanol (β-mercaptoethanol) ), 1% non-essential amino acids and antibiotics (penicillin/streptomycin), DMEM (Dulbecco's Modified Eagle'sMedia) culture solution was added at a volume ratio of 1:5, followed by a medical ultrasonic processor at a frequency of 40 kHz for 7 minutes. treatment to isolate the cells. And the isolated cells were obtained through centrifugation. And the obtained cells contained 10% Fetal Bovine Serum (FBS), 0.1 mM beta-mercaptoethanol, 1% non-essential amino acids and antibiotics (penicillin/streptomycin). Washed in DMEM (Dulbecco's Modified Eagle'sMedia) culture medium, suspended and cultured in a humidified chamber at _{37° C. and 5% CO 2 .} When the cells proliferated and the density increased on the surface of the culture dish, it was subcultured when it was almost confluent, and this time was designated as passage 1. The subculture process was repeated 5 times to obtain stem cells.

(Comparative Example 1)

Endometrial tissue was collected with a catheter according to the present invention. Then, it was washed with PBS (Phosphate Buffered Saline) under aseptic conditions, cut into pieces of 10 mm 3 or less, and treated with a collagenase solution (0.1% collagenase) at 37° C. for 60 minutes to separate single cell bodies. The separated cells were collected by filtration through a 100 μm nylon mesh. The recovered cells contained 10% Fetal Bovine Serum (FBS), 0.1 mM beta-mercaptoethanol, 1% non-essential amino acids and antibiotics (penicillin/streptomycin). It was washed in DMEM (Dulbecco's Modified Eagle's Media) culture medium, suspended and cultured in a humidified chamber at _{37° C. and 5% CO 2 .} When the cells proliferated and the density increased on the surface of the culture dish, it was subcultured when it was almost confluent, and this time was designated as passage 1. The subculture process was repeated 5 times to obtain stem cells.

(Test Example 1)

The properties of hEnMSCs were confirmed using fluorescence activated cell sorting analyzer (FACS). To analyze the surface antigen of hEnMSCs, cells were harvested with PBS containing 2 mM EDTA/5% FBS, and washed twice with PBS. Thereafter, the cells were placed in 2% FBS/PBS buffer, and an appropriate proportion of diluted fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibody (Becton, Dickinson and Company, CA, US) was added thereto at 4°C for 30 minutes. minutes were processed. 5 μg/ml of propidium iodide was added to remove dead cells and debris. Cell fluorescence was measured with FACS Vantage SE (Becton, Dickinson and Company, NJ, US), and the results were analyzed using the CellQuest (Becton, Dickinson and Company, NJ, US) program.

As a result of flow cytometry, Example 1 and Comparative Example 1 were all positive for CD44, CD73, CD90, CD105 and HLA-ABC, and negative for CD14, CD19, CD34, CD45, and HLA-DR. These results are consistent with the previously reported expression patterns of representative markers of mesenchymal stem cells (MSC).

(Experimental Example 2)

While the cells of Example 1 and Comparative Example 1 were cultured for 10 days, the number of cells between each group was counted for 10 days. The number of each cell was confirmed by trypan blue staining.

As a result, in Example 1, as the number of cells was gradually increased, good cell morphology was observed. On the other hand, in Comparative Example 1, the cell morphology was somewhat damaged, and the number of cells was increased to a level similar to that of Example 1.

As mentioned above, although the present invention has been described in detail using preferred embodiments, the scope of the present invention is not limited to specific embodiments, and should be interpreted by the appended claims. In addition, those skilled in the art should understand that many modifications and variations are possible without departing from the scope of the present invention.

100: insert 101: inlet
200: storage and discharge unit 201: outlet
210: stopper 220: separation means
221: through hole 300: removal string

Claims (5)

delete An insertion unit having a tubular shape and having at least one inlet through which the endometrial tissue and menstrual blood can flow into the rear of the open end;
a storage and discharge unit provided at the other open end of the insertion unit and formed with an outlet for discharging the introduced endometrial tissue and menstrual blood;
Includes a stopper for opening and closing the outlet,
The insertion unit is inserted into the uterus through the cervix of the woman,
The storage and discharge unit,
It is in the form of a balloon provided with an extension formed to surround a part of the other side of the insertion part,
The balloon-shaped storage and discharge unit is provided with a separation means connected to the other open end of the insertion unit therein,
The separation means is formed with a plurality of through-holes, endometrial tissue collection catheter, characterized in that for separating the endometrial tissue from the endometrial tissue and menstrual blood introduced through the insertion portion.
3. The method of claim 2,
On the outside of the balloon-shaped storage and discharge part,
Endometrial tissue collection catheter, characterized in that the removal string for removal of the catheter is further provided.
delete Collecting endometrial tissue using the catheter of claim 2 or 3;
Crushing the collected endometrial tissue (chopping);
ultrasonically treating the shredded endometrial tissue;
Method for obtaining stem cells from endometrial tissue, comprising the step of separating the cells by centrifuging the ultrasonically treated endometrial tissue.

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