A three-dimensional dermal papilla cell culture system and its construction method

Abstract

The present invention discloses a method for constructing a three-dimensional hair papilla cell culture system, comprising the following steps: S10, preparing a hair papilla cell suspension; S20, providing perfluorodecalin to prepare a liquid-liquid interface culture system of a perfluorodecalin-medium; S30, inoculating the hair papilla cell suspension into the liquid-liquid interface culture system to construct a three-dimensional hair papilla cell culture system. The present invention constructs a three-dimensional hair papilla cell culture system at a liquid-liquid interface, so that hair papilla cells can aggregate into a spherical three-dimensional structure in a soft matrix at the liquid-liquid interface, and the formed three-dimensional hair papilla cell microspheres can maintain their biological functions, providing effective support for tissue engineering skin research to achieve a large number of hair follicle regeneration.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a three-dimensional dermal papilla cell culture system and a construction method thereof. Background Art

[0002] Hair follicles are composed of epithelial components (hair follicle stem cells, matrix cells, outer root sheath cells and inner root sheath cells) and dermal components (hair papilla cells, dermal sheath cells). The hair papilla cells gather into balls to form the hair papilla structure of the hair follicle. The hair papilla is located in the hair bulb at the bottom of the hair follicle and is surrounded by matrix cells. As a special type of mesenchymal stem cells, hair papilla cells can regulate hair growth, hair cycle and hair follicle morphogenesis by communicating signals with matrix cells and hair follicle stem cells, and are the "headquarters" for hair follicle regeneration. The development and regeneration of hair follicles are the result of the interaction of signals between epithelial and dermal components. Hair papilla cells with hair follicle induction ability are one of the important seed cells for tissue engineering hair follicle reconstruction.

[0003] With the rapid development of tissue engineering and regenerative medicine in recent years, tissue engineering hair follicle reconstruction and stem cell therapy have become a promising direction for the treatment of androgenic alopecia. The principle of tissue engineering hair follicle reconstruction is to mix hair papilla cells with induction ability with epidermal stem cells in a certain proportion and then transplant them into the body, or to culture them in different ways in vitro to reconstruct new hair follicles. This method can regenerate more hair follicles by appropriately amplifying and mixing limited cells, which is expected to solve the dilemma of insufficient donor hair follicles in hair follicle transplantation. However, the number of hair papilla cells in hair follicles is scarce, and the number of primary cells and low-generation cells amplified by culture is difficult to meet the requirements of tissue engineering hair follicle reconstruction and stem cell therapy. During the process of in vitro amplification and culture, hair papilla cells will gradually lose their unique biological characteristics and functions, such as changing the behavior and morphology of cell growth, losing the protein expression of specific markers ALP, Versican, and Sox2, losing hair induction ability, and losing adult stem cell characteristics, which cannot meet the requirements of seed cells for tissue engineering hair follicle reconstruction and stem cell therapy. Therefore, it is currently difficult to obtain a sufficient number of dermal papilla cells with stemness and hair follicle induction ability through culture and expansion, which restricts the application of tissue engineering hair follicle reconstruction. Therefore, how to culture and expand dermal papilla cells in vitro and maintain their biological functions has become the key in the field of tissue engineering hair follicle regeneration.

[0004] Compared with traditional plastic, glass culture dishes and recently reported viscoelastic scaffolds, liquid-liquid interfaces (LLI) have various properties and present a semi-fluid environment. The present invention constructs an LLI culture system constructed with perfluorodecalin-complete culture medium. The protein film formed in the liquid-liquid interface can anchor living cells on a selected liquid interface, providing the soft interface that people are pursuing. This interface can dynamically adapt to the force generated by the cells, providing a new three-dimensional culture system for dermal papilla cells. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a three-dimensional hair papilla cell culture system and a three-dimensional hair papilla cell culture system constructed by the method, which has the biological characteristics of a normal hair papilla and can maintain the biological function of hair papilla cells in vitro.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] On the one hand, a method for constructing a three-dimensional dermal papilla cell culture system is provided. First, a liquid-liquid interface (LLI) culture system of perfluorodecalin-culture medium is prepared. In the LLI culture system, perfluorodecalin is located in the lower layer and the culture medium is located in the upper layer to form a soft liquid-liquid interface. The dermal papilla cell suspension prepared in advance is inoculated into the LLI culture system, and then the cells sink and anchor in the soft interface between the two layers of liquid, such as Figure 1 As shown, PFD in the figure is perfluorodecalin.

[0008] Specifically, the method for constructing a three-dimensional dermal papilla cell culture system comprises the following steps:

[0009] S10, prepare hair papilla cell suspension;

[0010] S20, providing perfluorodecalin to prepare a perfluorodecalin-culture medium liquid-liquid interface culture system;

[0011] S30, inoculating the hair papilla cell suspension into the liquid-liquid interface culture system to construct a three-dimensional hair papilla cell culture system.

[0012] The present invention constructs an LLI-type three-dimensional hair papilla cell culture system, so that the hair papilla cells can aggregate into a spherical three-dimensional structure in the soft matrix of the liquid-liquid interface, and the formed hair papilla cell microspheres can maintain their biological functions, providing effective support for tissue engineering skin research to achieve mass hair follicle regeneration.

[0013] As a preferred solution of the method for constructing a three-dimensional dermal papilla cell culture system, step S10 specifically includes the following steps:

[0014] S11, preparing the washed hair follicle unit, cutting the hair follicle at the upper end of the hair bulb under a stereoscope, and taking the hair bulb portion of the hair follicle;

[0015] S12, digesting the hair bulb so that the hair papilla is free from the hair bulb, and obtaining the hair papilla by centrifugation;

[0016] S13, placing the hair papilla in a culture dish containing culture medium, removing the culture, and subculturing to P3 to obtain the hair papilla cell suspension.

[0017] The follicle units are healthy follicle units obtained from the back of the head of patients with androgenic alopecia through FUE (Follicle unit extraction) hair follicle transplantation. The follicle units obtained through the operation are rinsed with PBS and then cut and separated to obtain the hair bulb.

[0018] As a preferred solution of the method for constructing a three-dimensional hair papilla cell culture system, in step S12, 0.1% neutral protease is used to digest the hair bulb of the hair follicle to separate the dermis and epidermis of the hair bulb. Specifically, after adding 0.1% neutral protease, the cells are placed in a 37°C incubator for 1-2 hours, and mixed by blowing every 15 minutes to separate the dermis and epidermis of the hair bulb.

[0019] As a preferred scheme for the construction of a three-dimensional hair papilla cell culture system, in step S12, the obtained dermal tissue is digested with a mixed enzyme consisting of 0.1% neutral protease and 0.2% collagenase. After adding the mixed enzyme, it is placed in a 37°C incubator for 1 hour, and is mixed by blowing every 15 minutes to allow the hair papilla to be free from the hair bulb.

[0020] As a preferred scheme for the construction method of a three-dimensional hair papilla cell culture system, in step S13, the hair papilla is migrated out and cultured using a DMEM culture medium containing 15% FBS (fetal bovine serum), and is subcultured to the P3 generation using a DMEM culture medium containing 10% FBS, and finally is digested with trypsin to obtain a P3 generation hair papilla cell suspension.

[0021] As a preferred solution of the method for constructing a three-dimensional dermal papilla cell culture system, step S20 specifically includes the following steps:

[0022] S21. Provide perfluorodecalin and a 24-well culture plate, and add perfluorodecalin to the bottom layer of the culture plate.

[0023] S22. Provide culture medium, gently add culture medium of equal volume to perfluorodecalin to the upper layer of perfluorodecalin, keep the culture plate stable during the whole process, let it stand for 5 minutes, and then culture it in a 37°C incubator to form a nanoprotein soft interface between the two layers of liquid, i.e., the perfluorodecalin-culture medium liquid-liquid interface culture system.

[0024] In step S22, the culture medium refers to DMEM complete culture medium containing 10% FBS. When culturing in a 37°C incubator, the complete culture medium is replaced once a day.

[0025] As a preferred scheme for the method of constructing a three-dimensional hair papilla cell culture system, step S30 is specifically: drop the hair papilla cell suspension on the upper layer of the LLI culture system of perfluorodecalin-culture medium. After standing, the hair papilla cells will sink to the middle layer of the perfluorodecalin-culture medium, thereby obtaining a three-dimensional hair papilla cell culture system.

[0026] On the other hand, a three-dimensional dermal papilla cell culture system constructed by the above construction method is provided, which can form a protein film at the liquid-liquid interface, thereby anchoring the dermal papilla cells on the soft interface formed by the LLI culture system.

[0027] The beneficial effects of the present invention are:

[0028] 1. The three-dimensional culture system constructed by the present invention can form a protein film at the liquid-liquid interface, thereby anchoring the dermal papilla cells on the soft interface. This interface can dynamically adapt to the force generated by the cells, allowing the dermal papilla cells to spontaneously aggregate into a three-dimensional structure of regular size.

[0029] 2. The present invention can effectively restore and maintain biological properties of hair papilla cells such as stemness and hair follicle induction ability, and provide support for seed cells required in the field of in vitro tissue engineering hair follicle reconstruction.

[0030] 3. The present invention is easy to operate, has low requirements on equipment and has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a general diagram and structural model diagram of the three-dimensional hair papilla cell culture system described in Example 1.

[0032] FIG. 2(A) is a diagram showing the initial state of adding the hair papilla cell suspension to the upper layer of the perfluorodecalin-medium as described in Example 1 (Bar=200 μm).

[0033] FIG2(B) is a state diagram of the dermal papilla cells described in Example 1 anchored on the liquid-liquid interface and spontaneously aggregated to form three-dimensional dermal papilla cell microspheres after being cultured on the perfluorodecalin-medium liquid-liquid interface for 48 hours (Bar=100 μm).

[0034] FIG3(A) is a diagram showing the initial state of adding the hair papilla cell suspension to the upper layer of the hexafluorobenzene-culture medium as described in Comparative Example 1 (Bar=200 μm).

[0035] FIG3(B) is a diagram showing the disintegration and necrosis of the hair papilla cells described in Comparative Example 1 after the cells were cultured on the hexafluorobenzene-medium liquid-liquid interface for 48 hours (Bar=200 μm).

[0036] FIG. 4(A) is a diagram showing the initial state of adding the hair papilla cell suspension to the upper layer of the perfluorooctane-medium as described in Comparative Example 2 (Bar=200 μm).

[0037] FIG4(B) is a diagram showing the disintegration and necrosis of the hair papilla cells described in Comparative Example 2 after the cells were cultured on the perfluorooctane-medium liquid-liquid interface for 48 hours (Bar=200 μm).

[0038] Figure 5 This is a fluorescence image of live-dead staining of three-dimensional dermal papilla cell microspheres described in Example 1 (Bar=50 μm).

[0039] Figure 6 It is a bar chart of the mRNA expression of biospecific markers of the three-dimensional dermal papilla cell microspheres and the two-dimensional cultured dermal papilla cells described in Example 1 (*P<0.05).

[0040] Figure 7 This is a Western blot result of the biological specific markers of the three-dimensional dermal papilla cell microspheres and the two-dimensional cultured dermal papilla cells described in Example 1. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further illustrated below through specific implementation methods.

[0042] Unless otherwise specified, various raw materials of the present invention can be purchased commercially or prepared according to conventional methods in the art.

[0043] In order to make the purpose, technical solution and advantages of the present invention clearer and more detailed, the technical solution of the present invention is further described in detail below in conjunction with implemented examples.

[0044] Various raw materials, equipment, consumables, etc. of the present invention can be purchased commercially, or prepared according to conventional experimental methods in the relevant technical field.

[0045] During the preparation process, aseptic operation must be strictly required, and the instruments and consumables used must be sterilized by high temperature and high pressure or gas disinfection.

[0046] Example 1

[0047] The construction method of the three-dimensional dermal papilla cell culture system of this embodiment is as follows:

[0048] (1) Cell source: Fresh follicular unit tissues from patients undergoing FUE autologous hair follicle transplantation were used. The extracted follicular units were rinsed with PBS and separated into individual follicles using microscissors under a stereoscope. The hair bulbs were cut off and digested with 0.1% neutral protease. The dermal and epidermal tissues of the follicles were then separated using microdissection under a stereoscope.

[0049] Preparation of complete medium: Add FBS to DMEM medium to make the concentration of FBS 10%.

[0050] Extraction and culture of hair papilla cells: Take the hair follicle dermis tissue obtained in the previous step, digest it with a mixed enzyme solution of 0.2% collagenase and 0.1% neutral protease for 1-2h (place it in a 37°C incubator for 1 hour, blow and mix it every 15 minutes to make the hair papilla free from the hair bulb), and add the same volume of complete culture medium to terminate the digestion process after digestion. Separate the hair papilla by differential centrifugation, resuspend it with PBS, and centrifuge it at 800-1500r / min for 3-5 minutes, remove the supernatant, add the complete culture medium to the hair papilla, and then transfer it to a culture dish, add 4ml of complete culture medium, place it in a cell culture incubator under 5% CO2 / 37°C conditions, and move it out for culture. Change the culture medium every 3 days. Then routinely subculture it to the P3 generation, digest the P3 generation hair papilla cells with trypsin, add the same volume of complete culture medium to terminate the digestion after digestion, discard the supernatant after centrifugation, and resuspend it with culture medium to obtain the P3 generation hair papilla cell suspension.

[0051] (2) Preparation of 24-well culture plates: Take out the 24-well cell culture plate and check the airtightness and integrity of the culture plate packaging. After disinfecting the surface of the packaging with alcohol, place it in a biosafety cabinet and disinfect it with ultraviolet light for 30 minutes. Open the package, take out the 24-well cell culture plate, open the culture plate cover, and turn it upside down on the table.

[0052] (3) In this embodiment, the bottom layer of the three-dimensional hair papilla cell culture system is made of perfluorodecalin, which is sterilized by filtration sterilization, and the filter is of Millex-GP 33mm PES 0.22um specification.

[0053] (4) Construction of a three-dimensional dermal papilla cell culture system: Use a pipette to add perfluorodecalin to a 24-well culture plate, keep the liquid level stable, then add complete culture medium and let it stand for 5 minutes. Use a pipette to add equal volumes of dermal papilla cell suspension to the upper layer of perfluorodecalin-culture medium. Keep the operation slow throughout the process. Then place the culture plate in a 5% CO2 / 37℃ cell culture incubator for culture. Change the culture medium once a day.

[0054] Comparative Example 1

[0055] This comparative example is basically the same as the above-mentioned Example 1, except that perfluorooctane is used as the bottom layer of the three-dimensional dermal papilla cell culture system.

[0056] Comparative Example 2

[0057] This comparative example is basically the same as the above-mentioned Example 1, except that hexafluorobenzene is used as the bottom layer of the three-dimensional dermal papilla cell culture system.

[0058] Test content:

[0059] The sphering of the hair papilla cells constructed in Example 1 and Comparative Examples 1-2 was observed, and live-dead staining was used to identify the survival of the cells in the hair papilla cell microspheres obtained by the system of the present invention, and the expression of markers related to the biological characteristics of the hair papilla cell microspheres was identified. The details are as follows:

[0060] The hair papilla cell suspensions in Example 1 and Comparative Examples 1-2 were inoculated into the LLI culture system, and observed and photographed under an inverted microscope to obtain Figures 2(A), 3(A), and 4(A), respectively. The hair papilla cell suspensions in Example 1 and Comparative Examples 1-2 were inoculated into the LLI culture system and cultured for 48 hours, and then observed and photographed under an inverted microscope to obtain Figures 2(B), 3(B), and 4(B), respectively. According to the experimental results, only when perfluorodecalin was used as the bottom liquid, the three-dimensional hair papilla cell microspheres could be successfully obtained. The three-dimensional hair papilla cell culture systems constructed with hexafluorobenzene and perfluorooctane as the bottom layer could not maintain the activity of the cells, which would lead to cell death.

[0061] The three-dimensional hair papilla cell microspheres constructed in Example 1 were subjected to live-dead staining to identify the survival of the cells in the microspheres. Observation under a fluorescence microscope revealed that the survival of the cells in the hair papilla cell microspheres was good. Figure 5 shown.

[0062] The three-dimensional hair papilla cell microspheres constructed in Example 1 were tested for gene expression of biological characteristic markers ALP, β-catenin, and Sox2 by qPCR, and compared with conventional two-dimensional cultured hair papilla cells (P3). The results showed that the mRNA expression of biological characteristic markers of the three-dimensional hair papilla cell microspheres was significantly higher than that of the two-dimensional cultured hair papilla cells. Figure 6 shown.

[0063] The three-dimensional dermal papilla cell microspheres constructed in Example 1 were used to detect the protein expression of biological characteristic markers ALP, β-catenin, and Sox2, and compared with the conventional two-dimensional cultured dermal papilla cells (P3). The results showed that the protein expression of biological characteristic markers of the three-dimensional dermal papilla cell microspheres was significantly higher than that of the two-dimensional cultured dermal papilla cells. Figure 7 shown.

[0064] The above embodiments are only used to illustrate the detailed methods of the present invention. The present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing a three-dimensional dermal papilla cell culture system, characterized in that: The following steps are involved: S10, prepare hair papilla cell suspension; S20, providing perfluorodecalin and a 24-well culture plate, adding perfluorodecalin to the bottom layer of the culture plate; providing a culture medium, adding a culture medium of equal volume to that of perfluorodecalin to the upper layer of the culture plate, placing the culture medium in a 37°C incubator after standing for 5 minutes, and preparing a perfluorodecalin-culture medium liquid-liquid interface culture system; the culture medium refers to a DMEM complete culture medium containing 10% FBS; S30, adding the hair papilla cell suspension to the upper layer of the liquid-liquid interface culture system of perfluorodecalin-culture medium, and allowing the hair papilla cell suspension to stand in the middle layer of the liquid-liquid interface culture system to obtain a three-dimensional hair papilla cell culture system.

2. The method for constructing a three-dimensional hair papilla cell culture system according to claim 1, characterized in that: Step S10 specifically includes the following steps: S11, preparing a hair follicle unit after washing with PBS, cutting the hair follicle at the upper end of the hair bulb under a stereoscope, and taking the hair bulb portion of the hair follicle; S12, digesting the hair bulb so that the hair papilla is free from the hair bulb, and obtaining the hair papilla by centrifugation; S13, placing the hair papilla in a culture dish containing culture medium, removing the culture, and subculturing to P3 to obtain a hair papilla cell suspension.

3. The method for constructing a three-dimensional hair papilla cell culture system according to claim 2, characterized in that: In step S12, the bulb portion of the hair follicle is digested with 0.1% neutral protease to separate the dermis and epidermis of the bulb portion; and the dermis is digested with a mixed enzyme consisting of 0.1% neutral protease and 0.2% collagenase to free the hair papilla from the bulb portion.

4. The method for constructing a three-dimensional hair papilla cell culture system according to claim 2, characterized in that: In step S13, the hair papilla is cultured out using a DMEM medium containing 15% FBS, subcultured using a DMEM medium containing 10% FBS, and finally digested with trypsin to obtain a P3 hair papilla cell suspension.

5. A three-dimensional dermal papilla cell culture system, characterized in that: It is prepared by the construction method described in any one of claims 1 to 4.

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