KR20210117985A - Follicle cell spheroid formed on substrate and method for preparing the same - Google Patents

Abstract

The present invention relates to hair follicle cell spheroids formed on a substrate and a manufacturing method thereof. The hair follicle cell spheroids formed on the substrate according to the present invention can effectively control the aggregation of superficial root sheath cells or keratinocytes on large-scale dermal papilla spheroids. In particular, by forming an array of adhesion patterns spaced apart at regular intervals on the substrate, in particular, a radial adhesion pattern array with a thin guide line, the hair follicle cell spheroids are concentrated in the center within individual adhesion patterns of the adhesion pattern array, and thus it is possible to maintain a certain interval between the hair follicle cell spheroids.

Description

Hair follicle cell spheroid formed on a substrate and method for manufacturing the same

The present invention relates to a hair follicle cell spheroid formed on a substrate and a method for preparing the same.

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, after forming a dermal papilla spheroid using these dermal papilla cells, aggregating the outer root sheath cells present in the outer root sheath of the hair follicle thereon, and then individually transplanting it is being performed. However, there have been difficulties in uniformly forming large-scale dermal papilla spheroids and controlling the aggregation of superficial root sheath cells thereon, 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.

The present invention is a substrate; an array of attachment patterns formed on the substrate and spaced apart from each other; dermal papilla spheroids formed on the attachment pattern array; and hair follicle cell spheroids formed on a substrate including superficial root sheath cells or keratinocytes formed by aggregation on the dermal papilla spheroids.

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 is a substrate; and an array of attachment patterns formed on the substrate and spaced apart from each other; dermal papilla spheroids formed on the attachment pattern array; And it provides a hair follicle cell spheroid formed on a substrate comprising the outer root sheath cells or keratinocytes formed by aggregation on the dermal papilla spheroids.

The outer root sheath cells or keratinocytes may be formed by aggregation in whole or in part on the surface of the dermal papilla spheroid.

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 central portion and the peripheral portion may be connected by at least one guide line.

The guide line may have a length of 100 μm to 500 μm, a thickness of the guide line may be 1 μm to 20 μm, and the number of guide lines may be 20 to 100.

The average diameter of the dermal papilla spheroids may be 100 μm to 500 μm.

In one embodiment of the present invention, (a) forming an array of attachment patterns spaced apart from each other on a substrate; (b) seeding and culturing dermal papilla cells on the adhesion pattern array to form dermal papilla spheroids; and (c) seeding and culturing superficial root sheath cells or keratinocytes on the substrate on which the dermal papilla spheroids are formed to aggregate them.

The ratio of the number of dermal papilla cells in step (a) and the superficial root sheath cells or keratinocytes in step (b) may be 5:1 to 1:5.

The hair follicle cell spheroids formed on the substrate according to the present invention can effectively control the aggregation of superficial root sheath cells or keratinocytes on large-scale dermal papilla spheroids.

In particular, by forming an adhesion pattern spaced apart at regular intervals on the substrate, in particular, a radial adhesion pattern array with a thin guide line, the hair follicle cell spheroids can be centrally located in the individual adhesion patterns of the adhesion pattern array. In addition, it is possible to maintain a certain interval between the hair follicle cell spheroids.

1 is a diagram illustrating a first radial attachment pattern of the present invention.
2A is a photograph showing a hair follicle cell spheroid formed on a substrate using a first radial adhesion pattern.
Figure 2b is a schematic diagram showing a hair follicle cell spheroid formed on a substrate using the first radial adhesion pattern. Keratinocytes in FIG. 2B refer to external root sheath cells or keratinocytes.
3 is a diagram illustrating a second radial attachment pattern of the present invention.
4A is a photograph showing a hair follicle cell spheroid formed on a substrate using a second radial adhesion pattern.
4B is a schematic diagram showing a hair follicle cell spheroid formed on a substrate using a second radial adhesion pattern. Keratinocytes in FIG. 4B refer to external root sheath cells or keratinocytes.
Figure 5 shows the results of confirming the shape of the dermal papilla-outer root sheath cell structure through individual cell fluorescence staining.
6 shows the results of confirming the proliferation of hair follicle epithelial cells in the dermal papilla-outer root sheath cell structure through fluorescence staining.
Figure 7a shows the shape of the first and second days of culture of the dermal papilla-keratinocyte cell construct.
Figure 7b shows the shape of the 3rd to 5th day of culture of the dermal papilla-keratinocyte cell construct.

The present inventors sequentially seeded dermal papilla cells and outer myoblast cells or keratinocytes on a patterned substrate while conducting a study to uniformly form large-scale dermal papilla spheroids and control the aggregation of outer myoblast cells or keratinocytes thereon. And by culturing, the present invention was completed.

Hair follicle cell spheroids formed on the substrate

The present invention is a substrate; an array of attachment patterns formed on the substrate and spaced apart from each other; dermal papilla spheroids formed on the attachment pattern array; And it provides a hair follicle cell spheroid formed on a substrate comprising the outer root sheath cells or keratinocytes formed by aggregation on the dermal papilla spheroids.

As used herein, the term "hair follicle cell spheroid" broadly refers to a mass in which hair follicle cells are aggregated, and in this case, the term "hair follicle cell" is a concept including dermal papilla cells and outer myoblast cells or keratinocytes present in the hair follicle.

The hair follicle cell spheroid formed on the substrate according to the present invention comprises: a substrate; an array of attachment patterns formed on the substrate and spaced apart from each other; dermal papilla spheroids formed on the attachment pattern array; And it comprises a superficial root sheath cells or keratinocytes formed by aggregation on the dermal papilla spheroids.

The attachment pattern array is formed on a substrate and characterized in that it is spaced apart at a predetermined interval, specifically, it is preferable to maintain a predetermined interval of 600 μm to 1,000 μm, and it is more preferable to maintain 800 μm to 1,000 μm. , but not limited thereto.

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.

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. More specifically, the length of the guide line may be 100 μm to 500 μm, preferably 200 to 400 μm, and more preferably 250 to 350 μm. The guide wire may have a thickness of 1 μm to 20 μm, and preferably 1 μm to 9 μm. The number of the guide lines may be 20 to 100, preferably 35 to 75. Thereby, the dermal papilla spheroids can be centrally located in the center within the individual attachment patterns. In particular, by optimizing the length, thickness, and number of guide lines, even when cells are seeded and cultured once, large-scale cell spheroids and superficial root sheath cells or keratinocytes can be uniformly formed.

For example, the individual attachment pattern may be preferably radial, and the thickness of the guide line may be preferably thin with a thickness of less than 10 μm.

The dermal papilla spheroids are characterized in that they are formed on the attachment pattern array in a three-dimensional form, 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 of the present invention is preferable in terms of being able to implement a size similar to that of human dermal papilla tissue.

The outer root sheath cells or keratinocytes are characterized in that they are formed by aggregation on the dermal papilla spheroids, have an orientation in the opposite direction to the substrate on the dermal papilla spheroids, and aggregation between the outer root sheath cells can be made. The outer root sheath cells or keratinocytes may be formed wholly or partially aggregated on the surface of the dermal papilla spheroid, and the outer root sheath cells or keratinocytes may be partially aggregated and formed on the surface of the dermal papilla spheroids. When the outer root sheath cells or keratinocytes are partially aggregated and formed on the surface of the dermal papilla spheroid, the contact area or contact location can be variously controlled.

On the other hand, the superficial root sheath cells or keratinocytes aggregated on the dermal papilla spheroids may be fixed in a hydrogel film.

Method for forming hair follicle cell spheroids on a substrate

In addition, the present invention (a) forming an array of attachment patterns spaced apart from each other on a substrate; (b) seeding and culturing dermal papilla cells on the adhesion pattern array to form dermal papilla spheroids; and (c) seeding and culturing superficial root sheath cells or keratinocytes on the substrate on which the dermal papilla spheroids are formed to aggregate them.

First, the method for forming hair follicle cell spheroids on a substrate according to the present invention includes forming an array of attachment patterns spaced apart at regular intervals on the substrate [step (a)]. Thereby, the dermal papilla cells can be seeded and cultured on the adhesion pattern array formed on the substrate.

Since the attachment pattern array is the same as described above, a redundant description thereof will be omitted.

Next, the method for forming hair follicle cell spheroids on a substrate according to the present invention includes the step of forming dermal papilla spheroids by seeding and culturing the dermal papilla cells on the adhesion pattern array [(b) step] .

The dermal papilla cells seeded on the adhesion pattern array may be 50,000 to 200,000 cells/well, and the culture may be performed at 30 to 40°C. In some cases, the seeding and culturing may be repeated two or more times, and in this case, the number of dermal papilla cells to be seeded per well may be divided by the number of repetitions.

Next, the method for forming hair follicle cell spheroids on the mother substrate according to the present invention includes the step of seeding and culturing superficial root sheath cells or keratinocytes on the substrate on which the dermal papilla spheroids are formed to aggregate them [(c) step] do.

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. The outer root sheath cells or keratinocytes may be formed wholly or partially aggregated on the surface of the dermal papilla spheroid, and the outer root sheath cells or keratinocytes may be partially aggregated and formed on the surface of the dermal papilla spheroid, but limited thereto. doesn't happen When the outer root sheath cells or keratinocytes are partially aggregated and formed on the surface of the dermal papilla spheroid, the contact area or contact location can be variously controlled.

In addition, the ratio of the number of the dermal papilla cells and the outer myoblast cells or keratinocytes may be 5:1 to 1:5, and the ratio of the numbers of the dermal papilla cells and the outer myoblast cells or keratinocytes is 2:1 to 1 It is preferably :1.

As described above, the hair follicle cell spheroids formed on the substrate according to the present invention include: a substrate; an array of attachment patterns formed on the substrate and spaced apart from each other; dermal papilla spheroids formed on the attachment pattern array; And it is characterized in that it comprises an outer root sheath cell or keratinocyte formed by aggregation on the dermal papilla spheroid, it is possible to effectively control the aggregation of the outer root sheath cell or keratinocyte on a large-scale dermal papilla spheroid.

In particular, by forming an array of attachment patterns (preferably, a radial attachment pattern; more preferably, a radial attachment pattern with a thin guide line) spaced apart from each other on the substrate, the hair follicle cell spheroids are separated from each other in the attachment pattern array. In addition to being able to focus on the central portion in the adhesion pattern, it is possible to maintain a certain interval between the hair follicle cell 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 radial attachment pattern array (guideline thickness: 10㎛)

<1-1> Preparation of the substrate on which the first radial attachment pattern array is formed

A first radial attachment pattern array with a center-to-center spacing of 800 µm was formed through patterning of a 96-well plate, a substrate (PAMCELL T001; ANK Co., Ltd.). At this time, the first radial attachment pattern (pattern diameter (guide wire length Х2): 600 μm, guide wire thickness and number: 10 μm and 36 pieces) 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 (DP cells) and outer root sheath cells (ORS 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.

First, 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 conditions.

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.

Next, ORS cells were grown and cultured on gibco 100 pie TCPS under conditions of Epilife culture medium (ORS culture medium) supplemented with 60 μm calcium, and then stored under liquid nitrogen conditions.

<1-3> Formation of large-scale dermal papilla spheroids through one-time seeding and culture

In (2), the DP cells proliferated through subculture were trypsinized for 3 minutes, dissolved in 10 ml of DP culture solution, 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 at the bottom in the DP culture medium, they were seeded at 100,000 cells/well on the substrate on which the first radial adhesion pattern array was formed in (1), and then cultured to obtain large-scale dermal papilla spheroids (DP spheroids). formed. Thereafter, it was stored in an incubator at 37°C.

<1-4> Aggregation of outer root sheath cells (ORS cells) through one-time seeding and culture

ORS cells stored under liquid nitrogen conditions in Example <1-2> were trypsinized for 3 minutes, dissolved in 10 ml of 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 ORS cells remaining on the bottom in the DP culture medium, it was seeded at 100,000 cells/well on the substrate on which large-scale dermal papilla spheroids were formed in (3), and then cultured. Thereafter, it was placed in an incubator at 37° C. and stored for 5 days, and photographs and schematic diagrams observing the results are shown in FIGS. 2(a) and (b).

Formation of dermal papilla spheroids using a radial attachment pattern array (guideline thickness: 3㎛)

A second radial attachment pattern array with a center-to-center spacing of 800 μm was formed through patterning of a 96-well plate, a substrate (PAMCELL T001; ANK Co., Ltd.). At this time, the second radial attachment pattern (pattern diameter (guide wire length×2): 600 μm, guide wire thickness and number: 3 μm and 36 pieces) is shown in FIG. 3 .

Then, in the same manner as in Example 1, dermal papilla spheroids were formed through seeding and culture once, and then, the outer root sheath cells (ORS cells) were aggregated through seeding and culture once (Fig. 4 (Fig. a) and (b)).

As shown in FIGS. 2(a) and 4(a), when a substrate on which a radial attachment pattern array is formed is used, large-scale DP spheroids with a diameter of about 150 to 350 μm are uniformly formed even when seeded and cultured once. , it is confirmed that ORS cells are formed in an aggregated state thereon. At this time, ORS cells are formed in a partially aggregated state on the large-scale DP spheroid surface.

On the other hand, as shown in Fig. 2(b), when the thickness of the guide line is thick (about 10 μm or more), like the first radial attachment pattern array, some DP cells and ORS cells along the guide line over time. It is confirmed that there is a problem that the agglomeration is dismantled. However, as shown in FIG. 4(b), when the thickness of the guide line is thin (less than about 10 μm), as in the second radial attachment pattern array, the aggregation of DP cells and ORS cells along the guide line even over time It is confirmed that it is not dismantled. Therefore, it is confirmed that most large-scale DP spheroids and ORS cells are concentrated in the center in the second radial attachment pattern.

Confirmation of the dermal papilla-extrinsic root sheath cell structure

<3-1> Shape analysis through individual cell identification

The shape of the dermal papilla-extrinsic root sheath cell structure prepared in <Example 2> was confirmed through individual cell fluorescence staining.

As a result, as shown in [Fig. 5], it was possible to confirm the hair follicle epithelial cells surrounding the dermal papilla tissue.

<3-2> Confirmation of proliferation of hair follicle epithelial cells

The proliferation of hair follicle epithelial cells in the dermal papilla-extrinsic root sheath cell structure prepared in <Example 2> was confirmed through fluorescence staining.

As a result, as shown in [Fig. 6], it was possible to confirm the area of the proliferated outer myoblast cells under the electron microscope.

Preparation of dermal papilla-keratinocyte cell structure

In the same manner as in <Example 2>, a dermal papilla-keratinocyte cell structure was prepared ( FIGS. 7a and 7b ).

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 (9)

Board;
an array of attachment patterns formed on the substrate and spaced apart from each other;
dermal papilla spheroids formed on the attachment pattern array; and
Containing superficial root sheath cells or keratinocytes formed by aggregation on the dermal papilla spheroids,
Hair follicle cell spheroids formed on the substrate.
According to claim 1,
The superficial root sheath cells or keratinocytes are hair follicle cell spheroids formed on a substrate, characterized in that they are formed by aggregation in whole or in part on the surface of the dermal papilla spheroid.
According to claim 1,
The individual attachment pattern of the attachment pattern array is divided into a central portion and a peripheral portion, and the central portion has a higher adhesion per unit area than the peripheral portion, a hair follicle cell spheroid formed on a substrate.
4. The method of claim 3,
The adhesion force per unit area is characterized in that controlled by the adhesion pattern density or adhesion pattern material, hair follicle cell spheroids formed on a substrate.
4. The method of claim 3,
The central and peripheral portions are characterized in that connected by at least one or more guide lines, hair follicle cell spheroids formed on a substrate.
6. The method of claim 5,
The length of the guide line is 100 μm to 500 μm, the thickness of the guide line is 1 μm to 20 μm, and the number of guide lines is 20 to 100, hair follicle cell spheroids formed on a substrate.
According to claim 1,
The average diameter of the dermal papilla spheroids is characterized in that 100 μm to 500 μm, hair follicle cell spheroids formed on a substrate.
(a) forming an array of attachment patterns spaced apart from each other on a substrate;
(b) seeding and culturing dermal papilla cells on the adhesion pattern array to form dermal papilla spheroids; and
(c) seeding and culturing superficial root sheath cells or keratinocytes on the substrate on which the dermal papilla spheroids are formed, and aggregating them
A method of forming hair follicle cell spheroids on a substrate.
9. The method of claim 8,
The method of forming hair follicle cell spheroids on a substrate, characterized in that the ratio of the number of dermal papilla cells in step (a) and the outer root sheath cells or keratinocytes in step (b) is 5:1 to 1:5.

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