WO2020116921A2

WO2020116921A2 - Dental pulp stem cell container

Info

Publication number: WO2020116921A2
Application number: PCT/KR2019/016977
Authority: WO
Inventors: 주경민; 남현; 이경민
Original assignee: (주)메디노; 성균관대학교산학협력단
Priority date: 2018-12-06
Filing date: 2019-12-04
Publication date: 2020-06-11
Also published as: KR102166804B1; WO2020116921A3; KR20200069089A

Abstract

The present disclosure relates to a container for storing a dental pulp stem cell and, more specifically, to a dental pulp stem cell container for efficiently obtaining a dental pulp stem cell from a tooth. A dental pulp stem cell container according to an embodiment of the present disclosure comprises: a container body (10) in which a cell stock solution is contained; a cover (20) coupled to the container body (10); a compound enzyme (30) received in the cover (20) to extract a dental pulp stem cell from a tooth; and a blocking film (40) for supporting the compound enzyme (30) to prevent the compound enzyme from being separated from the cover (20).

Description

Dimensional stem cell storage container

The present disclosure relates to a container for storing pulp stem cells, and more particularly, to a pulp stem cell storage container for efficiently obtaining pulp stem cells from teeth.

Unless otherwise indicated in this specification, the contents described in this identification item are not prior art to the claims of this application, and the description in this identification item is not admitted to be prior art.

The pulp stem cells are stem cells located in the teeth, and the pulp stem cells are extracted from the inside of the tooth. Dimensions are stored in solid teeth and contain large amounts of mesenchymal stem cells. The dimensional stem cells have a very high density, are excellent in cell proliferation and differentiation ability, and can be used for regenerative medicine not only to the owner of the tooth, but also to the parents and grandparents, so that the utilization value is considerably high. Recently, due to the advantage of being able to easily obtain mesenchymal stem cells from the pulp stem cells, even in the teeth that are naturally removed or removed by surgery, interest in use as a source of adult stem cells for teeth has increased (Republic of Korea Patent Registration). 10-1609335).

As described above, it is important to safely store and extract the dimensional stem cells for the treatment of various diseases. As a conventional method for extracting pulp stem cells from a tooth, 1) First, a defective site such as tooth decay or inflammation is removed from the extracted tooth, and then washed with PBS containing 1% penicillin-streptomycin. Here, PBS stands for Phosphate buffer saline (phosphate buffered saline), and the cell originally has a unique pH and osmotic concentration, which means a solution having a very similar pH and osmotic concentration. Therefore, washing with the PBS solution puts less burden on the cells, so many experiments use PBS for washing. 2) Next, 500 μl of Collagenase type 1 mg/mL was treated for enzymatic treatment, incubated for 15 minutes, and large tissues were removed using a 70 μm strainer, and only the supernatant was centrifuged at 1500 rpm and 3 min. It goes through a series of steps to measure the number of pulp stem cells.

However, in the case of the conventional method as described above, it is inefficient to extract the dimensional stem cells every time a tooth is extracted, so that the extracted tooth is collected until at least several dozens of teeth are collected in MEM alpha, PBS, HBSS. There is a disadvantage in that it takes a lot of time because it is stored under the temperature condition of 4°C for several days in the stalk stem cell storage solution. In addition, there is a disadvantage in that the number of pulp stem cells extracted is reduced because pulp stem cells may be damaged or lost in the process of extracting pulp tissue from the tooth.

The present invention has been filed to solve the problems in the prior art as described above, without having to store the extracted tooth in the dimensional stem cell storage solution for a long time, the dimensional stem cell storage container that can quickly and easily extract the dimensional stem cells The purpose is to provide.

In addition, it is obvious that other technical problems may be derived from the following description, not limited to the technical problems as described above.

Dimensional stem cell storage container according to an embodiment of the present disclosure, the container body 10 containing the cell storage solution, the cover 20 and the cover 20 to be coupled to the container body 10, the cover 20 of It is characterized in that it comprises a mixed enzyme (30) for extracting pulp stem cells from the teeth, and a blocking film (40) supporting the mixed enzyme (30) so as not to escape from the cover (20).

According to a preferred feature of the present disclosure, the cover 20 includes an outer coupling portion 210 coupled with an outer portion of the inlet of the container body 10 and an inner coupling coupled with an inner portion of the inlet of the container body 10. A portion 220 and a lower portion are opened, and the inner coupling portion 220 is mutually supported, and further includes a receiving portion 230 accommodating the mixed enzyme 30, and the blocking film 40 is the inner coupling portion (220) is characterized in that it is configured to be configured at the bottom to prevent separation of the mixed enzyme (30) accommodated in the receiving portion (230).

According to a preferred feature of the present disclosure, it is located on the top of the cover 20, and when pressed toward the lower portion, the blocking portion 40 is torn, and the pressurizing portion (30) is transferred to the mixing body 30 to the container body 10 ( 50).

According to a preferred feature of the present disclosure, the mixed enzyme 30 is characterized in that it is at least one selected from the group consisting of collagenase type I, DNase I, papain, cysteine and dispase.

According to a preferred feature of the present disclosure, the lower surface of the container body 10 is characterized in that it is formed in the shape of a cone 11 or a convex portion 13.

According to an embodiment of the present disclosure, unlike a method of extracting pulp stem cells at a time after storing several teeth at a time due to simple and simple storage or use of a culture container, tooth extraction is performed from multiple teeth. At the same time, it has the advantage of being time-efficient because it can extract pulp stem cells.

In addition, even if the pulp tissue is not extracted from the teeth by using forceps, the number of pulp stem cells that can be obtained increases because it can be extracted directly from the storage container.

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description of the claims.

1 is a photograph showing a process of separating pulp tissue from a conventional tooth.

2 is a perspective view of a dimensional stem cell storage container according to an embodiment of the present disclosure.

3 is a cross-sectional view of a dimensional stem cell storage container according to an embodiment of the present disclosure.

Figure 4 is a state of use of the dimensional stem cell storage container according to an embodiment of the present disclosure.

5 is a detailed view of another embodiment of the dimensional stem cell storage container according to an embodiment of the present disclosure.

Figure 6 is a detailed view of another embodiment of the dimensional stem cell storage container according to an embodiment of the present disclosure.

Figure 7 shows the number of cells and the time required to obtain the size of the stem cells and the time required for the storage of the size of the stem cells according to the present disclosure and the time required for the extraction of pulp stem cells according to the method for extracting the stem cells from the conventional teeth. Comparison graph.

Hereinafter, with reference to the accompanying drawings, look at the configuration, operation and effect of the dimension stem cell storage container according to a preferred embodiment. For reference, in the drawings, each component is omitted or schematically illustrated for convenience and clarity, and the size of each component does not reflect the actual size. In addition, the same reference numerals refer to the same components throughout the specification, and reference numerals for the same components in individual drawings will be omitted.

Figure 1 shows a photograph schematically illustrating the process of washing teeth in PBS and separating the dimensions from the teeth according to the conventional dimensional stem cell culture method.

One embodiment of a method for extracting dimensional stem cells from a conventional tooth is as follows. First, when a patient visits the dentist to extract a tooth or if the tooth naturally falls off the gum, it is stored in a storage tube and transported to the dental storage bank. Here, the storage tube is stored refrigerated at about 4°C, and the storage tube contains PBS (phosphate buffered saline) and an antibiotic to prevent teeth from being contaminated. Next, the pulp tissue is separated from the teeth with forceps, placed in a culture dish, and finely chopped with scissors. Thereafter, 500 μl of the enzyme Collagenase type I 1 mg/ml, which is required to extract pulp stem cells, is treated and cultured for 15 minutes. After that, a large tissue is removed using a 70 μm strainer, and only the supernatant is centrifuged at 1500 rpm and 3 min to obtain pulp stem cells. However, the above-described conventional dimensional stem cell extraction method causes a problem that many cells are damaged in the process of separating dimensional tissue from teeth using forceps, etc. The problem arises that the number of pulp stem cells obtained by extracting stem cells must be significantly reduced.

2 is a perspective view of a dimensional stem cell storage container according to an embodiment of the present disclosure, FIG. 3 is a sectional view, and FIG. 4 is a use state diagram.

Dimensional stem cell storage container according to the present disclosure is largely composed of a container body 10 and a cover 20 blocking the upper portion of the container body 10. Here, the cover 20 can be seen as a lid that serves to block the container from the external environment, and the container body 10 corresponds to a storage container for teeth containing cell storage liquid. The shape of the container body 10 may be configured as a general container shape in which a space is formed inside and all other areas except the open area are closed, and the cover 20 is an open area of the container body 10. It can be made of a configuration that can be closed.

The cell storage solution is not limited as long as it is a kind of medium or a buffer solution that is generally used, such as stem cell culture solution, buffer solution, and is, for example, MEM alpha, RPMI-1640, DMEM, PBS, HBSS, HEPES The MEM alpha may be used in various applications such as maturing human embryos into blastocysts, culturing functional organ tissues at micro-gravity, or culturing primary cells and cell lines. The container body 10 containing the cell storage solution is preferably blocked from outside air by the cover 20 at the top of the body. Here, the cover 20 may be mutually coupled to the container body 10 through a variety of coupling methods such as fitting and rotation, and the cell storage solution and teeth accommodated inside the container body 10 through such a combination are It serves to prevent it from escaping outside.

In the cover 20, a mixed enzyme 30 for extracting dimensional stem cells from teeth is accommodated therein. The mixed enzyme 30 corresponds to a dedicated enzyme for extracting pulp stem cells from teeth, preferably the mixed enzyme 30 is selected from the group consisting of collagenase type I, DNase I, papain, cysteine and dispase It is characterized by more than one. The mixed enzyme 30 in which the above components are mixed can be regarded as a mixed solution of components optimized to extract pulp stem cells from the teeth, and it can be prepared and used 10 times as concentrated, for example, the final treatment of the mixed enzyme The final concentration may be 1 μg to 10 mg, but the concentration of the enzyme generally used is not limited. The mixed enzyme 30 may be accommodated in the cover 20.

According to a preferred feature of the present disclosure, the cover 20 includes an outer coupling portion 210 coupled with an outer portion of the inlet of the container body 10 and an inner coupling coupled with an inner portion of the inlet of the container body 10. A portion 220 and a lower portion are opened, and the inner coupling portion 220 is mutually supported, and further includes a receiving portion 230 accommodating the mixed enzyme 30, and the blocking film 40 is the inner coupling portion (220) It is characterized in that it is configured to be configured at the bottom to prevent the mixed enzyme (30) accommodated in the accommodating portion (230) from being separated.

More specifically, the inner region of the cover 20 is the inlet region (or open region) of the container body 10, the outer coupling portion 210 for coupling with the outer portion 110 and the inlet region of the container body 10 It may include an inner coupling portion 220 for coupling with the inner portion 120. In one embodiment, when the water cap of the water bottle is rotated and coupled to the water bottle, similar to the method of mutually coupling the protrusion and the groove formed in a spiral, the outer coupling portion 210 is the outer portion 110 of the container body 10 ) And rotational coupling to achieve mutual coupling. In addition, the inner coupling portion 220 may be mutually coupled to the inner portion 120 of the container body 10 through a rotational coupling or a fitting coupling. Next, it further includes a receiving portion 230, the lower portion of which is opened to be mutually supported with the inner coupling portion 220 of the cover 20. The side portion of the accommodating portion 230 may be fitted to be mutually supported with the inner coupling portion 220, and the lower portion is configured to be open, so that when the blocking film 40 is later torn, the mixed enzyme 30 accommodated therein Is discharged to the bottom so that it can be mixed with the cell storage solution.

The blocking membrane 40 for preventing the separation of the mixed enzyme 30 accommodated in the receiving portion 230 is configured at the bottom of the inner coupling portion 220 is further configured. Here, the blocking film 40 may be composed of a thin film that does not tear or break unless a certain external force is applied, and the mixed enzyme 30 accommodated inside the receiving part 230 is a receiving part 230 ) It can be easily detached because the lower part is open, but it is not deviated to the outside by the blocking film 40 formed at the lower end of the inner coupling part 220.

Here, the pressing portion 50 is located on the upper portion of the cover 20, and more preferably, may constitute an upper region of the receiving portion 230. Therefore, when the upper region of the receiving portion 230 is pressed using a finger or the like, the mixing portion 30 is obtained by tearing the blocking film 40 while the receiving portion 230 descends along the inner coupling portion 220. Is introduced into the container body (10).

The container body 10 accommodates the cell storage solution and the teeth therein, and the shape of the teeth is generally formed with a conical or convex groove therein, and the dimension stem cells are generally included inside the groove. Therefore, as the shape of the lower surface of the container body 10 is changed to the shape of a cone 11 or a convex portion 13 to match the general tooth appearance, a smaller amount of the mixed enzyme 30 than the conventional size stem cells Even if mixed with the storage solution, since the conical groove inside the tooth and the lower surface of the container body 10 are easily contacted, it is possible to easily extract dimensional stem cells. As a result, since the amount of the mixed enzyme 30 required per storage container is reduced, the effect of cost reduction is recognized.

As described above, using the dimensional stem cell storage container, the teeth contained with the cell storage solution in the container body 10 are mixed with the mixed enzyme 30 introduced from the cover 20 and artificially mixed to better mix. You can apply force. Next, when the storage container containing the tooth is cultured in a 5% CO ₂ incubator under a temperature condition of 90% humidity and 37° C. for about 30 minutes, a sufficient number of dimensional stem cells can be extracted from the tooth.

Figure 7 shows the number of cells and the time required to obtain the size of the stem cells and the time required for the storage of the size of the stem cells according to the present disclosure and the time required for the extraction of pulp stem cells according to the method for extracting the stem cells from the conventional teeth. It is a comparison graph.

More specifically, when extracting pulp stem cells through the existing method, the average cell number of pulp stem cells extracted from 3 teeth was found to be about 1.9x10 ⁵ ±0.2, and the required time was 27±0.2 minutes. It took about. However, as a result of the experiment using the dimensional stem cell storage container according to the present disclosure, it was found to be about 6.7x10 ⁵ ±0.7, and the required time was confirmed within 1 minute.

As a result, by utilizing the above-described storage of the dimensional stem cells, when the dimensional stem cells are obtained, there is an advantage that a much larger number of dimensional stem cells can be obtained in a short time compared to the conventional method.

The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, but the embodiments described in the specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention and represent all technical spirits of the present invention. It should be understood that there may be various equivalents and modifications that can replace them at the time of application. Therefore, the above-described embodiments are to be understood as illustrative and not restrictive in all respects, and the scope of the present invention is indicated by the following claims rather than the detailed description, and the meaning and ranges of the claims It should be construed that any altered or modified form derived from the equivalent concept is included in the scope of the present invention.

Using the dimensional stem cell storage container according to the present invention, compared to the conventional method, it is possible to extract the dimensional stem cells quickly and easily, as well as to obtain a much larger number of dimensional stem cells in a short time, more efficient As a method to supply adult stem cells of teeth, it is expected to be useful for various treatments using adult stem cells.

[Description of codes]

10: container body 11: cone

13: convex portion 15: lower cap

20: cover 30: mixed enzyme

40: blocking film 50: pressing portion

110: outer part 120: inner part

210: outer coupling portion 220: inner coupling portion

230: accommodation section

Claims

A container body 10 containing a cell storage solution;

A cover 20 coupled with the container body 10;

A mixed enzyme (30) accommodated inside the cover (20) to extract pulp stem cells from the teeth; And

It characterized in that it comprises a; blocking membrane 40 for supporting the mixed enzyme 30 so as not to be separated from the cover 20, pulp stem cell storage container.
According to claim 1,

The cover 20,

An outer coupling part 210 coupled with an outer part of the inlet of the container body 10;

An inner coupling part 220 coupled with an inner part of the inlet of the container body 10;

The lower portion is opened, and the receiving portion 230, which is mutually supported with the inner coupling portion 220 to accommodate the mixed enzyme 30, further includes,

The blocking membrane 40 is configured at the lower end of the inner coupling portion 220, characterized in that to prevent the separation of the mixed enzyme 30 accommodated in the receiving portion 230, dimensional stem cell storage container.
According to claim 2,

Located on the top of the cover 20, when pressed toward the bottom, the blocking membrane 40 is torn while the mixing enzyme 30 to be delivered to the container body 10; 50 further comprising; dimensions Stem cell storage container.
According to claim 1,

The mixed enzyme (30) is characterized in that at least one selected from the group consisting of collagenase type I, DNase I, papain, cysteine, and dispase, pulp stem cell storage container.
According to claim 1,

The lower surface of the container body 10 is characterized in that it is made of a cone (11) or a convex portion (13) shape, dimensional stem cell storage container.