KR20210117983A - Method for preparing dermal papilla spheroid via repeat seeding and culturing dermal papilla cell - Google Patents

Abstract

The present invention relates to a method for manufacturing dermal papilla spheroids through repeated seeding and culturing dermal papilla cells, and to a large-scale dermal papilla spheroids manufactured therefrom. The large-scale dermal papilla spheroids manufactured by the manufacturing method of the present invention can be uniformly formed with an average diameter of about 100 to 500 μm. In particular, by forming an array of radial attachment patterns spaced apart at regular intervals on the substrate, the large-scale dermal papilla spheroids can be centrally located in the individual attachment patterns of the attachment pattern array, as well as can maintain a certain distance between the large-scale dermal papilla spheroids.

Description

Method for manufacturing dermal papilla spheroids through repeated seeding and culture of dermal papilla cells

The present invention relates to a method for preparing dermal papilla spheroids through repeated seeding and culture of dermal papilla cells and to large-scale dermal papilla spheroids prepared therefrom.

The dermal papilla cells exist at the base of the hair follicle and are responsible for hair growth and hair follicle cycle regulation by supplying oxygen and nutrients to the cells constituting the hair follicle. Therefore, when the proliferation of dermal papilla cells is promoted, hair becomes healthy, hair growth is promoted, and hair loss can be prevented. Because hair growth proceeds as the epithelial cells surrounding the dermal papilla divide to form a hair shaft, the dermal papilla cells play an important role in regulating the division of epithelial cells. In addition, in androgenetic alopecia, the site where male hormones act on hair follicles is also the dermal papilla, and dermal papilla cells play a very important role in hair growth.

For the treatment of hair loss, using these dermal papilla cells to form dermal papilla spheroids, and then to individually transplant them is being performed. However, there have been difficulties in uniformly forming large-scale dermal papilla spheroids from dermal papilla cells, and research is needed to solve this problem. Moreover, since a lot of time and effort are required for individual transplantation, research to reduce this is also required.

Accordingly, the present inventors have made diligent efforts to provide a method for large-scale formation of dermal papilla spheroids from dermal papilla cells. As a result, repeatedly seeding and culturing dermal papilla cells on a substrate on which an adhesion pattern array is formed at regular intervals. It was confirmed that dermal papilla spheroids could be prepared on a large scale, and the present invention was completed.

Accordingly, an object of the present invention is to (a) seeding and culturing dermal papilla cells at 20,000 to 100,000 cells/well on a substrate; And (b) repeating the step (a) two or more times to provide a method for producing a dermal papilla spheroid comprising the step of forming a dermal papilla spheroid.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention comprises the steps of (a) seeding and culturing dermal papilla cells on a substrate at 20,000 to 100,000 cells/well; and (b) repeating step (a) at least twice to form a dermal papilla spheroid.

Before step (a), the method may include forming an array of attachment patterns spaced apart from each other by a predetermined distance on the substrate.

The individual attachment patterns of the attachment pattern array may be divided into a central portion and a peripheral portion, and the central portion may have a higher adhesive force per unit area than the peripheral portion.

The adhesive force per unit area may be controlled by the density of the attachment pattern or the material of the attachment pattern.

The pattern may have a diameter of 200 to 1200 μm.

After step (b), seeding and culturing the superficial root sheath cells on the dermal papilla spheroids may further include aggregation.

In one embodiment of the present invention, it provides a dermal papilla spheroid, which is prepared by the above manufacturing method, and has an average diameter of 100 μm to 500 μm.

Large-scale dermal papilla spheroids prepared according to the manufacturing method of the present invention can be uniformly formed with an average diameter of about 100 μm to 500 μm. In particular, by forming an array of radial attachment patterns spaced apart at regular intervals on the substrate, the large-scale dermal papilla spheroids can be centrally located in the individual attachment patterns of the attachment pattern array, as well as between the large-scale dermal papilla spheroids. A certain distance can be maintained.

1 is a diagram showing a circular attachment pattern of the present invention.
Figure 2a is a photograph showing the dermal papilla spheroid formation process according to one-time repeated seeding and culture of dermal papilla cells using a circular adhesion pattern array.
Figure 2b is a photograph showing the dermal papilla spheroid formation process according to the repeated seeding and culture of dermal papilla cells using a circular adhesion pattern array twice.
Figure 2c is a photograph showing the dermal papilla spheroid formation process according to three times repeated seeding and culture of dermal papilla cells using a circular adhesion pattern array.
Figure 2d is a photograph showing the dermal papilla spheroid formation process according to the repeated seeding and culture of dermal papilla cells four times using a circular adhesion pattern array.
3 is a diagram illustrating a radial attachment pattern of the present invention.
Figure 4a is a photograph showing the dermal papilla spheroid formation process according to repeated seeding and culture of dermal papilla cells using a radial adhesion pattern array.
Figure 4b is a photograph showing the dermal papilla spheroid formation process according to repeated seeding and culture of dermal papilla cells using a radial adhesion pattern array.
Figure 4c is a photograph showing the dermal papilla spheroid formation process according to repeated seeding and culture of dermal papilla cells using a radial adhesion pattern array.
Figure 4d is a photograph showing the dermal papilla spheroid formation process according to repeated seeding and culture of dermal papilla cells using a radial adhesion pattern array.
5 shows the results of fluorescence staining for dermal papilla spheroids prepared using a radial adhesion pattern array.
Figure 6 is a photograph showing the dermal papilla spheroid formation process according to one-time seeding (200,000 cells/well) and culture of dermal papilla cells in a radial adhesion pattern array.
7a shows the results of dermal papilla spheroid formation according to the pattern diameter (200, 400 or 600 μm) of the radial attachment pattern array.
7b shows the results of dermal papilla spheroid formation according to the pattern diameter (800, 1000 or 1200 μm) of the radial attachment pattern array.
8 shows the results of dermal papilla spheroid formation according to the thickness of the guide line (3 or 10 μm) to the number of guide lines (36, 72 or 144) of the radial attachment pattern array.

The present inventors completed the present invention by using a substrate and repeatedly seeding and culturing dermal papilla cells while conducting a study on a method for uniformly forming large-scale dermal papilla spheroids from dermal papilla cells.

Accordingly, the present invention comprises the steps of (a) seeding and culturing dermal papilla cells at 20,000 to 100,000 cells/well on a substrate; And (b) repeating the step (a) two or more times may provide a method for producing a dermal papilla spheroid comprising the step of forming a dermal papilla spheroid.

That is, in step (a), the number of dermal papilla cells seeded once on the substrate is 20,000 to 100,000 cells/well, preferably 30,000 to 70,000 cells/well.

The incubation may be performed for 3 to 5 hours at a temperature of 30 to 40°C.

In step (b), the number of repetitions of step (a) is 2 or more, preferably 2 to 8 times, and more preferably 3 to 5 times.

According to a preferred embodiment of the present invention, the method for manufacturing the dermal papilla spheroids may include the step of forming an array of attachment patterns spaced apart at regular intervals on the substrate before the step (a). Thereby, the dermal papilla cells can be seeded and cultured on the adhesion pattern array formed on the substrate.

According to a preferred embodiment of the present invention, the attachment pattern array is characterized in that it is spaced apart from each other at a predetermined interval on the substrate. Specifically, the predetermined interval is preferably maintained at 600 to 1,000 μm, more preferably 800 μm. It is preferable to maintain the thickness of 1,000 μm.

The attachment pattern array means a plurality of arrangement of individual attachment patterns.

Specifically, the individual attachment pattern may be divided into a central portion and a peripheral portion based on a 1/2 point at a distance from the center to the circumference, and the central portion may have a higher adhesive force per unit area than the peripheral portion. In this case, the adhesion force per unit area can be adjusted by the adhesion pattern density or the adhesion pattern material. High adhesion pattern material can be applied.

The individual attachment patterns may be circular or radial, preferably radial.

The diameter of the pattern may be 200 to 1200 μm, preferably 300 to 1000 μm, and more preferably, the interval may be 300 to 800 μm.

In particular, the central portion and the peripheral portion may be connected by one or more guide lines. In this case, the length, thickness, and number of the guide lines may be variously adjusted. Thereby, the dermal papilla spheroids can be centrally located in the center within the individual attachment patterns.

The thickness of the guide wire may be 1 to 20 μm, preferably 1 to 9 μm.

The number of the guide lines may be 20 to 100, preferably 35 to 75.

In addition, the method for producing dermal papilla spheroids according to the present invention may further include, after step (b), aggregating by seeding and culturing outer root sheath cells on the dermal papilla spheroids.

The outer root sheath cells have an orientation in the opposite direction to the substrate on the dermal papilla spheroid, and aggregation between the outer root sheath cells may be made.

On the other hand, the superficial root sheath cells aggregated on the dermal papilla spheroid can be fixed in the hydrogel film.

According to a preferred embodiment of the present invention, it is possible to provide a three-dimensional dermal papilla spheroids having an average diameter of 100 μm to 500 μm, prepared by the method for producing the dermal papilla spheroids. The diameter of the dermal papilla spheroids may preferably have an average diameter of 150 μm to 350 μm, more preferably 180 μm to 250 μm. The dermal papilla spheroid manufacturing method of the present invention is preferable in terms of being able to implement a size similar to that of human dermal papilla tissue.

As described above, the method for producing dermal papilla spheroids according to the present invention comprises the steps of (a) seeding and culturing dermal papilla cells at 20,000 to 100,000 cells/well on a substrate; and (b) repeating step (a) two or more times to form large-scale dermal papilla spheroids having an average diameter of about 100 μm to 500 μm uniformly. can

In particular, by forming an array of attachment patterns (preferably, radial attachment patterns) spaced apart at regular intervals on the substrate, the large-scale dermal papilla spheroids can be centrally located within the individual attachment patterns of the attachment pattern array, as well as , it is possible to maintain a certain interval between the large-scale dermal papilla spheroids.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Formation of dermal papilla spheroids using a circular attachment pattern array

<1-1> Preparation of substrate on which circular attachment pattern array is formed

Through patterning of a 96-well plate, a substrate (PAMCELL T001; ANK Co., Ltd.), a circular attachment pattern array with a spacing between the centers of 800 μm was formed. At this time, a circular attachment pattern (pattern diameter: 400 μm) is shown in FIG. 1 . At this time, the material of the substrate is a non-cell-adhesive material, which is formed of polyethylene glycol (PEG) hydrogel, and the patterning uses cell-adhesive peptide-coated particles, but UV rays are locally applied through a photomask. It was carried out in a manner of exposure (exposure).

<1-2> Preparation of dermal papilla cells

After collecting hair follicles during the hair follicle transplantation process, hair follicles that could not be transplanted were selected, and dermal papilla cells (DP cells) and outer root sheath cells (ORS cells) were isolated. At this time, DP cells were proliferated and cultured on gibco 100 pie TCPS under hyclone/low glucose culture medium (DP culture medium) supplemented with 10% FBS and 1% anti-anti, and then stored under liquid nitrogen condition.

Thereafter, the DP cells stored under liquid nitrogen conditions were subcultured. Specifically, DP cells stored under liquid nitrogen conditions were dissolved in 10 ml of DP culture medium, and then put into a tube. After the tube was treated in a centrifuge at 1300 rpm for 3 minutes, the supernatant was removed. The DP cells remaining at the bottom were dispersed in the DP culture medium and divided into three gibco 100 pie TCPSs for proliferation and culture. After about 3 days, DP cells were removed by trypsinizing each TCPS for 3 minutes, and then treated in a centrifuge at 1300 rpm for 3 minutes, and then the supernatant was removed. The DP cells remaining at the bottom were dispersed in the DP culture medium and divided into three gibco 100 pie TCPSs for proliferation and culture. Through this, a total of 9 gibco 100 pie TCPS cells were proliferated and cultured DP cells were obtained.

<1-3> Large-scale dermal papilla spheroid formation through repeated seeding and culture

The DP cells proliferated through subculture in Example <1-2> were trypsinized for 3 minutes, dissolved in 10 ml of a DP culture medium, and put into a tube. After the tube was treated in a centrifuge at 1300 rpm for 3 minutes, the supernatant was removed. After dispersing the DP cells remaining on the bottom in the DP culture medium, they were seeded at 50,000 cells/well on the substrate on which the circular adhesion pattern array was formed in (1), and then cultured. Thereafter, it was placed in an incubator at 37° C. and stored for 4 hours, and a photograph observing the results is shown in FIG. 2( a ). The above process was repeated a total of 4 times to form a large-scale dermal papilla spheroid (DP spheroid), and the results were observed each time it was repeated.

As a result, as shown in [Fig. 2a] to [Fig. 2d], when the DP cells were repeatedly seeded and cultured 4 times at 50,000 cells/well on the substrate on which the circular adhesion pattern array was formed, as the number of repeated seeding was repeated, the DP It was confirmed that the spheroids were uniformly formed as the average diameter of the spheroids increased. Finally, it was confirmed that large-scale DP spheroids with an average diameter of about 150 to 350 μm were uniformly formed. However, it was confirmed that some large-scale DP spheroids were not concentrated in the center of the circular attachment pattern.

Formation of dermal papilla spheroids using a radial attachment pattern array

<2-1> Formation of dermal papilla spheroids using a radial attachment pattern array

Through patterning of a 96-well plate, a substrate (PAMCELL T001; ANK Co., Ltd.), a radial attachment pattern array with a spacing between the centers of 800 μm was formed. In this case, the radial attachment pattern (pattern diameter (guide wire length×2): 400 μm, guide wire thickness and number: 10 μm and 72 pieces) is shown in FIG. 3 . Then, large-scale dermal papilla spheroids (DP spheroids) were formed through repeated seeding and culture in the same manner as in Example 1.

As a result, as shown in [Fig. 4a] to [Fig. 4d], even when DP cells are repeatedly seeded and cultured 4 times at 50,000 cells/well on the substrate on which the radial adhesion pattern array is formed, the number of repeated seeding increases It was confirmed that the DP spheroids were uniformly formed as the average diameter increased, and finally, it was confirmed that large-scale DP spheroids with an average diameter of about 150 to 350 μm were uniformly formed. In particular, it was confirmed that most of the large-scale DP spheroids were concentrated in the center within the radial attachment pattern.

<2-2> Fluorescence check

With respect to the DP spheroids formed in Example <2-1>, it was attempted to confirm the expression of versican through fluorescence staining.

Specifically, the DP spheroids formed on the substrate were immobilized with paraformaldehyde, and 8 μm frozen sections were prepared, followed by staining with a primary antibody selectively binding to versican protein. Thereafter, fluorescence immunostaining was completed using a secondary antibody with green fluorescence, and the fluorescence image was confirmed through a confocal fluorescence microscope.

As a result, as shown in , DP spheroids were formed, and it was confirmed that versican was also expressed.

[Comparative Example 1]

The DP cells proliferated through subculture in Example <1-2> were trypsinized for 3 minutes, dissolved in 10 ml of a DP culture medium, and put into a tube. After the tube was treated in a centrifuge at 1300 rpm for 3 minutes, the supernatant was removed. After dispersing the DP cells remaining on the bottom in the DP culture medium, they were seeded at 200,000 cells/well on the substrate on which the radial adhesion pattern array was formed in (1), and then cultured. Thereafter, it was placed in an incubator at 37° C. and stored for 16 hours.

As a result, as shown in [Fig. 6], when DP cells are seeded and cultured once at 200,000 cells/well on a substrate on which a radial adhesion pattern array is formed, DP spheroids are not formed uniformly, and neighboring DPs are not formed. It was confirmed that agglomeration between spheroids occurred.

Confirmation of conditions for dermal papilla spheroid formation using a radial attachment pattern array

Instead of forming the dermal papilla spheroids in the same manner as in Example <2-1>, the diameter of the radial attachment pattern (guide line length × 2) was 200, 400, 600, 800, 1000 or 1200 μm; The guide wire thickness is 3 or 10 μm; The number of guide lines was set to 144, 72 or 36 to form and compare dermal papilla spheroids.

The results are shown in [Fig. 7a] to [Fig. 8].

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (7)

(a) seeding and culturing dermal papilla cells at 20,000 to 100,000 cells/well on a substrate; and
(b) repeating step (a) two or more times to form a dermal papilla spheroid
Method for preparing dermal papilla spheroids.
According to claim 1,
Before the step (a), the method of manufacturing a dermal papilla spheroid comprising the step of forming an array of attachment patterns spaced apart at regular intervals on a substrate.
3. The method of claim 2,
The individual attachment pattern of the attachment pattern array is divided into a central portion and a peripheral portion, and the central portion is characterized in that the adhesion per unit area is higher than that of the peripheral portion, the method of manufacturing a dermal papilla spheroid.
4. The method of claim 3,
The adhesion force per unit area is characterized in that controlled by the adhesion pattern density or the adhesion pattern material, the manufacturing method of the dermal papilla spheroids.
3. The method of claim 2,
The pattern has a diameter of 200 to 1200 ㎛, characterized in that the dermal papilla spheroid manufacturing method.
According to claim 1,
After the step (b), the method for producing a dermal papilla spheroid, further comprising the step of seeding and culturing the outer root sheath cells on the dermal papilla spheroids to aggregate.
The dermal papilla spheroids prepared by the manufacturing method according to claim 1, having an average diameter of 100 μm to 500 μm.

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