TWI733035B - Dynamic three-dimensional cell sphere culture method and cell sphere, and multi-layer folliculoid cell sphere culture method and multi-layer folliculoid cell sphere for hair formation - Google Patents

Abstract

Embodiments disclose a dynamic three-dimensional cell culture method, a cell sphere, a multi-layer folliculoid cell sphere culture method and a multi-layer folliculoid cell sphere for growingin vitro a multi-layer cell sphere similar to a hair follicle structure. The multi-layerfolliculoid cell sphere is cultured by sequentially adding dermal papilla cells, keratinocytes and adipose stem cells in a three-dimensional cell culture environment, in the steps of: the dermal papilla cells forming a spheroid; coating an outer surface of the spherical body withthe keratinocytes to form a first layer of outer shell; and coating the adipose stem cells outside the first layer of outer shell to form a second layer of outer shell. The multi-layerfolliculoid cell sphere has a layered structure similar to that of a hair follicle. The disclosed embodiments expect to help regenerate the hair follicles in vitro and provide a more suitable models for hair loss research. The present invention effectively increases the hair follicle formation rate in intended applications and use.

Description

Dynamic three-dimensional cell culture method and cultivation of cell ball and hair follicle-like multilayered cell ball Nourishing method and hair follicle-like multilayered cell ball

The present invention belongs to a dynamic three-dimensional cell culture method and cell ball, hair follicle-like multilamellar cell spheroid culture method and hair follicle-like multilamellar cell ball, which are derived from dermal papilla cells obtained from C57BL/6 mice. Cells, DP cells), keratinocytes (keratinocytes, KCs) and adipose-derived stem cells (ASCs) are co-cultured with three-dimensional hair follicle-like multilamellar cell spheres, which can mimic the structure of hair follicles in the body. ability.

According to statistics from the American Society for Hair Loss Research in 2016, the number of people with symptoms of hair loss in the world has exceeded 1 billion, and the causes of hair loss are numerous, such as family inheritance, severe trauma, chronic diseases, life stress and so on. Hair loss not only affects appearance and even confidence, but also reduces physiological functions, such as preventing heat loss and preventing ultraviolet radiation. In recent years, many studies have pointed out that through appropriate cultivation methods and applications, hair regeneration can be seen in mouse animal experiments, thus showing that stem cell therapy has considerable development potential for the treatment of hair loss.

There are many treatments for hair loss, but there are still some limitations. Oral drugs or topical medications must be used for a long time, usually accompanied by side effects; hair transplants are expensive, and the transferred hair follicles will still face the possibility of shrinking, which usually needs to be used with topical medications; laser hair treatment technology is not stable, and may cause Hair follicle damage has a considerable degree of risk. The above method is still not In the application of regenerative medicine, it is a feasible research direction to invent a method to regenerate hair follicles through physiological research or cell therapy.

At present, more studies have pointed out that after appropriate differentiation induction and three-dimensional culture of mouse pluripotent stem cells, lamellar cell aggregates at the embryonic stage can be obtained. The mesoderm is enclosed by the ectoderm, and finally It will form a skin organ, which can be used as a good in vitro model for future scientists to study the regeneration of appendages on the skin (Jiyoon Lee, et al. 2018). Therefore, how to simulate the structure of hair follicles in vitro for research should be another direction of development.

The patent case of Invention No. 201440762 "A pharmaceutical composition for improving the activity of hair follicle mesenchymal stem cells and promoting hair growth and its production method" announced on November 1, 103 of the Republic of China is a patent case that promotes the growth of hair follicles and its cells. An active pharmaceutical composition. The pharmaceutical composition in this case contains flavonoid derivatives and adenosine compounds, especially catechin derivatives and adenosine, as active ingredients, and a cosmetically or pharmaceutically acceptable carrier. In a preferred embodiment, the pharmaceutical composition of this case further includes a culture derived from hair follicle mesenchymal stem cells. By using the pharmaceutical composition of the invention, hair growth can be promoted.

Since the previous patent case only focuses on improving the activity of hair follicle mesenchymal stem cells, combining specific compounds can promote hair growth, but there is no clear in vitro model for its application to prove its application, and it lacks animal experiments or clinical experiments. The function of this method is only similar to the existing external drugs, and it is not an ideal technique for hair follicle regeneration.

There is also a patent case of Invention No. I585108 "Peptide for Promoting Hair Growth" published on June 1, 106 of the Republic of China, which discloses: a peptide is composed of amino acid sequences and is derived from organisms. It is extracted and obtained through digestion treatment, chemical synthesis, or genetic engineering, and it is used to cultivate dermal papillary cell proliferation promoters.

The previous patent case only focuses on promoting the proliferation of dermal papillary cells, and has not been used in animal experiments or clinical trials, and has not provided further descriptions on how to rebuild hair follicles. It is also not an ideal hair regeneration technology.

There is also a patent case of Invention No. 201717992 "A composition for promoting hair growth or hair cell growth and its use" announced on June 1, 106 of the Republic of China, which discloses: a kind of promoting hair growth or promoting hair cell growth A composition comprising a multi-peptide composition and a fat-soluble compound, wherein the fat-soluble compound is adiponectin or vitamin D3; the volume percentage of the multi-peptide composition is 1% to 20% of the multipeptide In the composition, the adiponectin is 0.1 nanogram per milliliter (ng/ml) to 100 ng/ml, and the vitamin D3 has a concentration of 1 nanomolar (nM) to 1000 nM. The invention also provides a use of the aforementioned composition for culturing a medicine that promotes hair growth or hair cell growth, wherein an effective dose of the medicine is administered to a recipient to promote hair growth or hair cell growth in the recipient , In order to solve the problem of ineffectiveness of the existing drugs for the treatment of hair loss.

The previous patent application uses different compounds to synthesize medicines that can promote hair growth or hair cell growth. It is not the application of regenerative medicine, and the current medicine level is not an ideal research direction for hair follicle regeneration.

Therefore, after evaluation, the inventor of the present invention used the technical content previously obtained with the Republic of China Invention Patent No. 14903375 to disclose the use of chitosan (Chitosan) to suspend and aggregate cells in culture to form cell spheres. Methods to develop related research that can simulate the spherical structure of hair follicles, thereby providing a better in vitro model for hair loss research or as a direction to promote hair follicle regeneration.

Therefore, the inventor once proposed the Republic of China Invention Patent No. I601820 "Medicinal composition for promoting hair follicle formation and its preparation method and use", which used dermal papillary cells and fat cells to form a nucleocapsid sphere to simulate the structure of hair follicles. However, this structure is relatively simplified, and the actual situation of simulating hair follicle growth in vitro will be greatly different, so there is still room for improvement.

In view of this, in view of the current known methods of studying hair loss, there is still considerable room for development. Therefore, the present invention provides a hair follicle-like multilamellar cell ball for simulating the composition of hair follicles and further hair regeneration. This hair follicle-like multilamellar cell ball system is cultured using a dynamic three-dimensional cell culture method: providing a three-dimensional cell culture environment, Combine the three-dimensional cell culture environment with the planar oscillation system, and then place dermal papilla cells (DP cells), keratinocytes (KCs) and adipose-derived stem cells (ASCs) in sequence The three-dimensional cell culture environment is co-cultured under suspension and rotation, and the multilamellar cell spheres of this kind of hair follicle covered by layers are formed to simulate the structure of hair follicles, and from the inside to the outside, dermal papilla cells (dermal papilla cells) are formed. ,DP cells), keratinocytes (KCs) and adipose-derived stem cells (ASCs).

The present invention further provides a method for culturing hair follicle-like multilayered cell spheres, which includes the following steps: Step (a) provides a dermal papillary cell, the dermal papillary cell is cultured in a three-dimensional cell culture environment, and the three-dimensional cell culture environment can be dynamically three-dimensional cell culture, so that the dermal papillary cell forms a spheroid by itself And step (b) providing a keratinocyte to join the three-dimensional cell culture environment, so that the keratinocytes are coated outside the spheroid to form a first shell; and step (c) providing an adipose stem cell to join the three-dimensional cell culture environment , So that the adipose stem cells are coated outside the first layer of shell to form a second layer of shell, thereby forming a type of hair follicle multilamellar cell sphere with three layers of cells, and furthermore can also produce more than three layers of multilamellar cells Ball (multi-layer sphere). .

The above-mentioned dynamic three-dimensional cell culture method involves moving a planar oscillator into the cell culture box, and then placing a petri dish that provides a three-dimensional cell culture environment on the planar oscillator and rotating it to achieve three-dimensional cell culture under dynamic conditions. .

In the above-mentioned dynamic three-dimensional cell culture method, the culture temperature of the three-dimensional cell culture environment is between 32°C and 42°C, and the culture environment air system adds carbon dioxide with a volume percentage of between 3% and 7% in the air, and the culture time is intermediate. Between 5 days and 7 days, and the oscillation parameters are between 80 rpm and 120 rpm. Preferably, the rotational speed of the planar oscillator is set to 100 rpm, and the cell incubator parameters are set to 37° C., 5% carbon dioxide, and kept in the whole process of cell culture for 6 days without interruption.

In the above-mentioned dynamic three-dimensional cell culture method, the three-dimensional cell culture environment is to coat chitosan with a weight percentage between 0.8% and 1.2% on a culture medium to form a chitosan film, and then the dermis Papillary cells, keratinocytes and adipose stem cells are sequentially placed on the chitosan film. Further, in the three-dimensional cell culture environment, 1% by weight of chitosan is coated on the medium base, and the medium base is a chassis with a plurality of sharp holes, such as a 96-hole pan.

The above-mentioned hair follicle-like multilamellar cell line was taken from dermal papillary cells, keratinocytes and adipose stem cells of C57BL/6 mice, among which dermal papillary cells and adipose stem cells were from C57BL/6 mice aged six to eight weeks. , Keratinocytes come from C57BL/6 newborn mice within three days after birth.

The culturing process of the above-mentioned hair follicle-like multilayered cell ball is as follows. It is manufactured by sequential inoculation. The culture medium is 445 ml of high-sugar medium (Dulbecco's Modified Eagle's Medium, DMEM) containing 50 ml of fetal bovine serum (fetal bovine). Serum, FBS) and 5 ml of penicillin and streptomycin (P/S). First, on day 0, dermal papillary cells were seeded ^{with 150 microliters of culture medium per well containing 3x10 4 cells.} a chassis having a tip 96 of chitosan film culture and in a plane shaker for 2 days to form a dermal papilla cell pellet; on day 2 to 50 [mu] l keratinocyte culture medium per well containing 2.5x10 ⁴ th Several kinds of cells are put into the culture plate. Under dynamic conditions, the keratinocytes will coat the first formed dermal papillary cell spheres in the pores of the dermal papillary cell spheres, and cultured for 2 days, the cells will be covered evenly and completely; on the 4th day, the adipose stem cells will be divided into each well. 50 microliters of culture medium containing 2.5x10 ⁴ cells are seeded into the culture plate. Under dynamic conditions, the adipose stem cells will coat the double-layered cell spheres in the pores. The inner layer is the dermal papillary cell sphere, and the outer layer is the outer layer. Then it is the first shell composed of keratinocytes; the second layer of adipose stem cells can be evenly and completely coated outside the first shell to form a multi-layered cell sphere with three layers of different cell populations. .

The dynamic three-dimensional cell culture method of the present invention can also culture other cell spheres composed of different cells as required.

The above technical features have the following advantages:

1. The three-dimensional cell culture under the 96-point well chassis can retain the hair-inducing ability of dermal papillary cells. Each hole forms a cell sphere independently and is uniform in size. At the same time, if there is contamination, it will only occur in that specific hole. Will not affect the cell pellets in other wells.

2. Hair follicles are derived from the interaction between epithelium and mesenchyme, and are divided into epidermis and dermis with the specialization of the skin after birth. The close contact between the two and related signal molecules The transmission makes the dermal papilla gradually differentiate and is covered by specialized epidermal cells to form hair follicles, and the hair follicles are surrounded by subcutaneous fat tissue that can secrete relevant growth factors. Therefore, according to the structural composition of real hair follicles, the three main cell groups that make up hair follicles are based on the order of mesenchymal dermal papillary cells as the inner layer, epithelial keratinocytes as the first shell, and adipose stem cells as the second shell. A multi-layered cell sphere is cultivated so that the multi-layered cell sphere can simulate the composition of hair follicles in the body.

3. The dynamic culture environment can make the aggregation of the three kinds of cells more uniform and thorough, and the formed cell aggregate can be closer to the sphere and closer to the original spherical shape of the hair follicle under the skin.

(A): Chitosan film

(B): Cell

(C): 96-well tip tray with cells (with chitosan film)

(D): Planar oscillator

(E): Hair follicle-like multilayered cell ball

(F): Cell incubator

(G): Dermal papillary cells of hair follicle-like multilamellar cell balls

(H): Keratinocytes of hair follicle-like multilamellar spheres

(I): Adipose stem cells of hair follicle-like multilamellar spheres

(J): Specialized keratinocytes of real hair follicles

(K): The dermal papilla of real hair follicles

(L): Subcutaneous fat tissue

[Figure 1A] is a schematic diagram of a dynamic three-dimensional cell culture device according to an embodiment of the present invention.

[The first image B] is an enlarged view of the sharp hole in the 96 sharp hole chassis coated with a chitosan film in the first image A.

[Second Picture A] is a schematic diagram of real hair follicle structure.

[Second Figure B] is a schematic diagram of the multilamellar sphere of hair follicles of the present invention.

[Third Figure A] is a flow chart of the cultivation of the hair follicle multilamellar cell spheres according to the embodiment of the invention.

[Third Figure B] is a micrograph of the multilamellar cell spheres of hair follicles in an embodiment of the present invention.

[Fourth Figure] is a schematic diagram of the fluorescence signs of frozen sections of the multilayered structure of the hair follicle-like multilayered cell sphere of the present invention.

Please refer to Figure 1A and Figure 1B, the embodiment of the present invention includes:

A dynamic three-dimensional cell culture device in which a 1% w/v chitosan solution is coated on a 96-well tip chassis (C) to form a 96-well tip chassis with a chitosan film (A) (C), the chitosan solution applied is made from chitosan powder (85% deacetylation degree; Sigma-Aldrich), and 5 grams of the powder is dissolved in 495 ml of ultra-pure Mix water and 5 ml of glacial acetic acid for 24 hours to prepare a 1% w/v chitosan solution. Each well of the 96-well tip bottom plate (C) must be cultured in a certain step after applying 180 microliters of chitosan solution. It should be dried at 70°C for at least 6 hours, and then passed through an equivalent concentration of 1N hydroxide. Inject 180 microliters of sodium (NaOH) into each hole for acid-base neutralization reaction for 1 minute, inject 180 microliters of phosphate buffered saline into each hole, and wash twice for 30 seconds each time, and inject 180 microliters of ultrafiltration into each hole. Wash with pure water twice for 30 seconds each time, put it in a 70℃ oven and dry for at least 30 minutes, and finally go through a 15-minute UV exposure treatment process to prepare a 96-hole tip chassis with a chitosan film ( C).

Place the cultured cells (B) on the 96-well tip chassis with chitosan film (C), and then move a flat shaker (D) to a 37°C, 5% carbon dioxide cell incubator (F) , Then apply A 96-well tipped chassis (C) with a chitosan film is placed on a plane shaker and rotated to achieve three-dimensional cell culture under dynamic conditions. The rotation speed of the plane shaker is set to 100rpm (E), and start The whole process of cell pellet culture is 6 days without interruption. Among them, the use of sharp holes for culture, due to the limitation of the shape of the sharp holes, will help the cells develop into spheres.

The layered design of this type of hair follicle multilamellar cell ball is comparable to the structure of real hair follicles. For example, the second picture A shows the structure of real hair follicles. The dermal papillary cells (K) covered by specialized keratinocytes (J) are located in the center of the hair follicle bulb, while the hair follicles are surrounded by subcutaneous fat tissue (L). As shown in Figure 2B, this type of hair follicle multilamellar cell sphere (E), the cell source is derived from the dermal papillary cells (G), keratinocytes (H) and adipose stem cells (I) of C57BL/6 mice, among which the dermal milk The dendritic cells and adipose stem cells were derived from C57BL/6 mice aged six to eight weeks, and the keratinocytes were derived from C57BL/6 newborn mice within three days of birth.

As shown in Figures 3A and 3B, the culture process of this type of hair follicle multilayered cell sphere (E) is as follows, which is manufactured by sequential cell seeding (M), and the culture used is The culture medium is 445 ml high glucose medium (Dulbecco's Modified Eagle's Medium, DMEM) containing 50 ml fetal bovine serum (FBS) and 5 ml penicillin and streptomycin (P/S). ), firstly, on day 0, dermal papillary cells (G) were seeded into a 96-well tip chassis (C) with a chitosan film (A) with ^{150 microliters of culture medium per well containing 3×10 4 cells and placed in} Culture on a flat shaker for 2 days to form a sphere of dermal papillary cells (G); on the second day, keratinocytes (H) are seeded into the culture plate ^{with 50 microliters of culture medium per well containing 2.5x10 4 cells} , Under dynamic conditions, keratinocytes (H) will coat the spheres of dermal papillary cells (G) formed in its pores, and culture for 2 days to coat them evenly and completely; on the 4th day, adipose stem cells (I ) Use 50 microliters of culture medium per well containing 2.5x10 ⁴ cells into the culture plate. Under dynamic conditions, the adipose stem cells (I) will coat the double-layered cell spheres in the wells, where the inner layer is dermal milk Globules (G), and the first shell is keratinocytes (H); the adipose stem cells (I) can be evenly and completely covered by culturing 2 days to form a second shell outside the first shell, and then form a A multi-layered cell sphere with three layers of different cell populations.

This type of hair follicle multilamellar cell ball (E) was received and embedded for frozen section on the 6th day of the culture process. Since the keratinocytes were first marked with DiO (green fluorescent cell stain; Biotium) before inoculation, the DiO It binds to the cell membrane of keratinocytes for tracking use; and adipose stem cells are labeled with DiI (red fluorescent cell stain; Invitrogen) before inoculation, so that DiI binds to the cell membrane of adipose stem cells for tracking use. The above-mentioned frozen section processing and sample embedding are all methods of tissue section analysis that can be understood by those with ordinary knowledge in the technical field of the present invention, and the method flow will not be described in detail here.

Please refer to the fourth figure. After this type of hair follicle multilayered cell sphere is frozen sectioned, use mounting medium with DAPI (DAPI-containing mounting glue; Abcam) for mounting and staining the nucleus of the cell spheroid. The fluorescent markers under a light microscope show the distribution of three different layers of cells. Among them, dermal papillary cells only show blue fluorescence (DAPI), while keratinocytes show green fluorescence (DiO), and adipose stem cells show red fluorescence. Light (DiI), so blue, green and red fluorescence should be visible from the inside out, showing multi-layered cell spheres. The above-mentioned fluorescent dyeing and other technologies are common methods that can be understood by those with ordinary knowledge in the technical field of the present invention, and the method flow will not be described in detail here.

Among them, the white circle in the 20-magnification superimposed picture is the 60-magnification group picture, and the distribution of the three-layer structure can be clearly seen through the larger magnification. Therefore, it is feasible to cultivate this kind of hair follicle multilamellar cell spheres by this dynamic three-dimensional cell culture method.

Based on the description of the above embodiments, when one can fully understand the operation and use of the present invention and the effects of the present invention, but the above embodiments are only the preferred embodiments of the present invention, and the implementation of the present invention cannot be limited by this. The scope, that is, simple equivalent changes and modifications made according to the scope of the patent application of the present invention and the content of the description of the invention, are all within the scope of the present invention.

(E): Hair follicle-like multilayered cell ball

(G): Dermal papillary cells of hair follicle-like multilamellar cell balls

(H): Keratinocytes of hair follicle-like multilamellar spheres

(I): Adipose stem cells of hair follicle-like multilamellar spheres

Claims (7)

A method for culturing hair follicle-like multilamellar cell spheroids in sequence includes the following steps: step (a) culturing dermal papillary cells in a three-dimensional cell culture environment, so that the dermal papillary cells form a spherical shape by themselves Body; and step (b) adding a keratinocyte to the three-dimensional cell culture environment, so that the keratinocyte is coated outside the spheroid to form a first shell; and step (c) adding an adipose stem cell to the three-dimensional cell culture Environment, so that the adipose stem cells are coated outside the first shell to form a second shell, thereby forming a type of hair follicle multilayered cell sphere; among them, it is manufactured by sequential cell seeding, and the The culture medium is 445 ml high-sugar medium containing 50 ml fetal bovine serum and 5 ml penicillin/streptomycin double antibiotics. First, on day 0, 150 microliters of dermal papillary cells per well contain 3x10 ⁴ cells. Into a 96-well tip chassis with a chitosan film and culture on a flat shaker for 2 days to form a sphere of dermal papillary cells; on the second day, the keratinocytes were divided into 50 microliters of culture medium containing 2.5x10 per well. ⁴ cells are seeded into the culture plate. Under dynamic conditions, keratinocytes will coat the spheres of dermal papillary cells that are formed in the pores, and culture for 2 days to coat them evenly and completely; on the 4th day, the adipose stem cells will be covered Use 50 microliters of culture medium per well containing 2.5x10 ⁴ cells into the culture plate. Under dynamic conditions, the adipose stem cells will coat the double-layered cell spheres in the pores, and the inner layer is the dermal papillary cell spheres. The first layer of shell is keratinocytes; a second layer of adipose stem cells can be covered uniformly and completely outside the first layer of shell and form a second layer of shell outside the first layer of shell. The method for culturing multilamellar cell spheres of hair follicles as described in item 1 of the scope of patent application, wherein the three-dimensional cell culture environment is oscillated to form a dynamic three-dimensional cell culture environment. The method for culturing hair follicle multilamellar cell balls as described in item 2 of the scope of patent application, wherein the culture temperature of the three-dimensional cell culture environment is between 32°C and 42°C, and the culture environment air system adds volume to the air The percentage is between 3% and 7% of carbon dioxide, the cultivation time is between 5 and 7 days, and the oscillation parameter is between 80 rpm and 120 rpm. The method for culturing hair follicle multilamellar cell spheres as described in item 3 of the scope of patent application, wherein the oscillation parameters are 100 rpm, culture temperature 37°C, culture environment air system with 5% carbon dioxide added to the air, and culture The time is 6 days. The method for culturing multilamellar hair follicle cell spheres as described in item 4 of the scope of patent application, wherein the three-dimensional cell culture environment is coated with chitosan with a weight percentage between 0.8% and 1.2%. A chitosan film is formed on the culture substrate, and then the dermal papillary cells, keratinocytes and adipose stem cells are sequentially placed on the chitosan film. The method for culturing hair follicle multilamellar cell spheres as described in item 5 of the scope of patent application, wherein the three-dimensional cell culture environment is coated with 1% by weight of chitosan on the substrate, and the culture The base is a chassis with multiple sharp holes. A hair follicle-like multilayered cell sphere is used as a composition for improving hair follicle regeneration. The hair follicle-like multilayered cell sphere includes: a dermal papillary cell in the form of a spheroid; a keratinocyte coated outside the spheroid A first shell is formed; and a fat stem cell is coated outside the first shell to form a second shell.

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