A method for isolating and culturing autologous dermal papilla cells

Abstract

The present invention relates to the technical field of separation and culture of autologous hair papilla cells, specifically to a method for separation and culture of autologous hair papilla cells, comprising the following steps: S1, sample preparation, taking a skin tissue sample from a target individual, selecting a hair-dense area on the top of the head, using a scalpel and laser equipment to remove hair follicles, using a micro blade and micro scissors to remove the skin tissue surrounding the hair follicles, i.e., the epidermis and part of the dermis, leaving only the hair follicle structure; S2, microfluidic channel design, designing a microfluidic chip, setting a plurality of microchannels in the chip, and coating the inner surface of the channel with extracellular matrix molecules that bind to hair papilla cells, i.e., collagen and fibronectin, to enhance cell adhesion. By combining physical and mechanical methods with biological sorting technology, higher separation efficiency and cell viability are obtained, mainly through microfluidic technology and electrostatic sorting, by precisely controlling the flow environment and electric field effect of hair follicle cells, efficient separation of autologous hair papilla cells is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of separation and culture of autologous dermal papilla cells, in particular to a separation and culture method of autologous dermal papilla cells. Background Art

[0002] Hair follicle stem cells (HFSCs) are relatively primitive cells in hair follicles. They are mesenchymal stem cells with strong self-renewal and multidirectional differentiation abilities. They are located in the outermost layer of the bulge of the outer root sheath of the hair follicle, close to the attachment point of the arrector pili muscle. Hair follicle stem cells can not only differentiate into hair follicles and effectively treat hair loss, but also participate in the formation of epidermal tissue. At the same time, they have strong immunomodulatory effects, hematopoietic support, and promote tissue repair.

[0003] The isolation and culture methods of autologous dermal papilla cells (DPCs) usually involve isolating and purifying these cells from hair follicles. Existing technologies mostly rely on enzymatic hydrolysis, microtubule cell separation or sorting methods based on surface markers;

[0004] The above method has low separation efficiency during the specific operation process, and the cell activity is easily affected and reduced.

[0005] Therefore, a method for isolating and culturing autologous dermal papilla cells is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for isolating and culturing autologous hair papilla cells, which achieves higher separation efficiency and cell viability by combining physical and mechanical methods with biological sorting technology. It mainly uses microfluidics technology and electrostatic sorting to accurately control the flow environment and electric field of hair follicle cells to achieve efficient separation of autologous hair papilla cells, so as to solve the problems raised in the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for isolating and culturing autologous dermal papilla cells, comprising the following steps:

[0008] S1. Sample preparation: take skin tissue samples from the target individual, select the hair-dense area on the top of the head, use a scalpel and laser equipment to remove the hair follicles, use a micro blade and micro scissors to remove the skin tissue around the hair follicles, that is, the epidermis and part of the dermis, leaving only the hair follicle structure;

[0009] S2. Microfluidic channel design. A microfluidic chip is designed. Multiple microchannels are set in the chip. The inner surface of the channel is coated with extracellular matrix molecules that bind to hair papilla cells, namely collagen and fibronectin, to enhance cell adhesion. The flow rate of the fluid is controlled so that the hair follicles are mechanically separated at a specific position when passing through the channel. The hair papilla cells are separated from other cells of the hair follicle through the pores of the microchannel. When the flow rate is v=10μm / s, the outer diameter of the hair follicle is 200-300μm, and the average diameter of the cells is 10μm-15μm, the hair papilla cells will preferentially attach to the chip surface.

[0010] S3. Electric field sorting: introducing an alternating electric field or a direct electric field into the microfluidic channel, and by applying the principle of Dielectrophoresis and utilizing the polarization response of different cells to the electric field, the selective separation of hair papilla cells is achieved. The movement rate of hair papilla cells in the electric field is related to their dielectric constant. The dielectric constant of hair papilla cells will be repelled by the negative electric field. The electric field force formula is: Where r is the cell radius, m is the electric constant of the medium, K(ω) is the dielectric function of the cell, and E is the electric field strength. By adjusting the electric field strength and frequency, the hair papilla cells move to a specific position under the action of the electric field, thereby achieving their separation;

[0011] S4, collecting and culturing, collecting the separated hair papilla cells into a preset culture dish or culture bottle through a microfluidic device, and using a customized culture medium containing growth factors, namely FGF and IGF, to promote the proliferation of the hair papilla cells;

[0012] S5. Cell verification: The purity of the isolated cells was verified by immunofluorescence staining and Western blot detection of hair papilla cell-specific markers, namely CD34 and ALP. Anti-CD34 antibody was used for staining to observe whether there was a DPCs-specific fluorescence signal and to detect the expression of the hair papilla cell marker ALP.

[0013] Preferably, said S3, electric field sorting, includes optimizing the relationship between electric field strength and frequency;

[0014] Among them, low frequency enhances the cell aggregation effect by increasing the electric field strength;

[0015] The high-frequency regulation of the electric field frequency causes "virtual separation" of cells, and the separation effect from other cells is maximized by adjusting the frequency.

[0016] Preferably, the specific electric field formula is as follows:

[0017]

[0018] in, represents the frequency-dependent dielectric function, E is the electric field intensity, r is the cell radius, and m is the dielectric constant.

[0019] Preferably, the low frequency stage is optimized, and at low frequency, the hair papilla cells show positive DEP. At this time, the migration effect of the hair papilla cells is enhanced by increasing the electric field strength, and the electric field strength is set to 1 kV / m-5 kV / m.

[0020] Preferably, the high-frequency stage is optimized. Under high frequency, the polarization effect of cells is weakened. At this time, the frequency of the electric field is adjusted.

[0021] Preferably, the S3, electric field sorting also includes frequency modulation and electric field waveform design, and the electric field frequency is modulated into a variable frequency so that the hair papilla cells show different migration characteristics at different frequencies, and different waveforms such as sine wave, square wave or triangle wave are used. These waveforms will affect the response intensity and directionality of the cells to the electric field when adjusting the electric field frequency. By optimizing the waveform form, the sorting accuracy can be further improved.

[0022] Preferably, the optimization analysis is performed using a mathematical model between electric field strength and frequency. When the electric field strength and frequency have a relationship with the cell migration speed, the following relationship is established:

[0023]

[0024] Among them, v cell is the migration speed of the cell, μ is the mobility of the cell in the electric field, E is the electric field strength, is the real part of the dielectric function.

[0025] Preferably, the S3, electric field sorting, includes precisely controlling the electric field distribution, by precisely controlling the arrangement, spacing and shape of electrodes to achieve a more uniform and stable electric field distribution.

[0026] Preferably, the S3, electric field sorting includes alternating use of multiple frequencies, alternating adjustment of low frequency and high frequency, using frequency scanning technology, gradually increasing from low frequency to high frequency, mobilizing the negative and positive DEP characteristics of hair papilla cells, and optimizing the sorting process.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] By combining microfluidics and electric field sorting, this method is efficient, precise and gentle in isolating hair papilla cells, and can avoid the cell damage that may be caused by traditional enzymatic hydrolysis. Subsequent optimization will further improve its separation efficiency and cell activity, providing reliable technical support for hair regeneration research and clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 It is the overall structural view of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] See also Figure 1 , the present invention provides a technical solution:

[0033] A method for isolating and culturing autologous dermal papilla cells comprises the following steps:

[0034] S1. Sample preparation: take skin tissue samples from the target individual, select the hair-dense area on the top of the head, use a scalpel and laser equipment to remove the hair follicles, use a micro blade and micro scissors to remove the skin tissue around the hair follicles, that is, the epidermis and part of the dermis, leaving only the hair follicle structure;

[0035] S2. Microfluidic channel design. A microfluidic chip is designed. Multiple microchannels are set in the chip. The inner surface of the channel is coated with extracellular matrix molecules that bind to hair papilla cells, namely collagen and fibronectin, to enhance cell adhesion. The flow rate of the fluid is controlled so that the hair follicles are mechanically separated at a specific position when passing through the channel. The hair papilla cells are separated from other cells of the hair follicle through the pores of the microchannel. When the flow rate is v=10μm / s, the outer diameter of the hair follicle is 200-300μm, and the average diameter of the cells is 10μm-15μm, the hair papilla cells will preferentially attach to the chip surface.

[0036] S3. Electric field sorting: introducing an alternating electric field or a direct electric field into the microfluidic channel, and by applying the principle of Dielectrophoresis and utilizing the polarization response of different cells to the electric field, the selective separation of hair papilla cells is achieved. The movement rate of hair papilla cells in the electric field is related to their dielectric constant. The dielectric constant of hair papilla cells will be repelled by the negative electric field. The electric field force formula is: Where r is the cell radius, mis the electric constant of the medium, K(ω) is the dielectric function of the cell, and E is the electric field strength. By adjusting the electric field strength and frequency, the hair papilla cells move to a specific position under the action of the electric field, thereby achieving their separation;

[0037] S4, collecting and culturing, collecting the separated hair papilla cells into a preset culture dish or culture bottle through a microfluidic device, and using a customized culture medium containing growth factors, namely FGF and IGF, to promote the proliferation of the hair papilla cells;

[0038] S5. Cell verification: The purity of the isolated cells was verified by immunofluorescence staining and Western blot detection of hair papilla cell-specific markers, namely CD34 and ALP. Anti-CD34 antibody was used for staining to observe whether there was a DPCs-specific fluorescence signal and to detect the expression of the hair papilla cell marker ALP.

[0039] Specifically, the S3, electric field sorting, includes optimizing the relationship between electric field strength and frequency;

[0040] Among them, low frequency enhances the cell aggregation effect by increasing the electric field strength;

[0041] The high-frequency regulation of the electric field frequency causes "virtual separation" of cells, and the separation effect from other cells is maximized by adjusting the frequency.

[0042] Specifically, the specific electric field formula is as follows:

[0043]

[0044] in, represents the frequency-dependent dielectric function, E is the electric field intensity, r is the cell radius, and m It is the dielectric constant of the medium. By selecting the appropriate frequency ω, the hair papilla cells can show different movement trends relative to other hair follicle cells.

[0045] Specifically, the low-frequency stage is optimized. At low frequencies, i.e., 10kHz-100kHz, the hair papilla cells show positive DEP. At this time, the migration effect of the hair papilla cells is enhanced by increasing the electric field strength, and the electric field strength can be set to 1kV / m-5kV / m. Since the polarization characteristics of hair papilla cells and other cells in this frequency band are different, the hair papilla cells can be effectively attached to the selected area of ​​the microfluidic channel by adjusting the electric field strength.

[0046] Specifically, the high-frequency stage is optimized. At high frequencies (100kHz to 1MHz), the polarization effect of the cells is weakened, which may be manifested as negative DEP. At this time, the response of the hair papilla cells should be distinguished from other cells as much as possible. At this time, by adjusting the electric field frequency to between 100kHz and 1MHz, the relative migration speed of the cells will change significantly. Therefore, a lower electric field strength (e.g., 500V / m-1000V / m) can be used at high frequencies to avoid damaging the cells and ensure the separation effect of the cells.

[0047] Specifically, the S3, electric field sorting also includes frequency modulation and electric field waveform design, modulating the electric field frequency to a variable frequency (for example: using a continuous conversion from low frequency to high frequency), so that the hair papilla cells show different migration characteristics at different frequencies, using different waveforms such as sine waves, square waves or triangle waves, which will affect the intensity and directionality of the cell's response to the electric field when adjusting the electric field frequency. By optimizing the form of the waveform, the sorting accuracy can be further improved.

[0048] Specifically, the mathematical model between electric field strength and frequency is used for optimization analysis. Assuming that the electric field strength and frequency have a relationship with the migration speed of cells, the following relationship can be established:

[0049]

[0050] Among them, v cell is the migration speed of the cell, μ is the mobility of the cell in the electric field, E is the electric field strength, is the real part of the dielectric function.

[0051] By adjusting EE and ω, accurate sorting of dermal papilla cells can be achieved. For example, in the low frequency band, the dielectric response of dermal papilla cells is large, and the migration speed can be enhanced by adjusting the electric field strength. In the high frequency band, adjusting the frequency can make other cells respond weaker, thereby enhancing the sorting effect.

[0052] Specifically, the S3, electric field sorting includes precisely controlling the electric field distribution, and precisely controlling the arrangement, spacing and shape of the electrodes to achieve a more uniform and stable electric field distribution. In particular, when a microelectrode array is used, a more precise electric field gradient can be generated, thereby further improving the sorting efficiency of hair papilla cells. In the case of non-uniform electric field strength, the electric field strength gradient can be adjusted by optimizing the electrode spacing and the distribution of the current source to ensure the effective separation and collection of hair papilla cells.

[0053] Specifically, the S3 electric field sorting includes using multiple frequencies alternately, using low frequency and high frequency to alternately adjust to avoid cross-interference between other hair follicle cells and hair papilla cells. For example, using frequency sweeping technology, gradually increasing from low frequency to high frequency, mobilizing the negative and positive DEP characteristics of hair papilla cells, and optimizing the sorting process.

[0054] Fluid flow model in microfluidic channels

[0055] Assume that the flow of hair follicle cells in the microfluidic channel obeys the Navier-Stokes equation, the flow velocity is vv, the channel length is LL, the channel cross-sectional area is AA, and it flows under a certain pressure gradient. According to the fluid dynamics formula:

[0056]

[0057] Where: Re is the Reynolds number, ρ is the fluid density, and μ is the fluid viscosity.

[0058] When the Reynolds number is low (eg, less than 2000), the flow is laminar and the flow rate can be stably controlled, facilitating cell separation.

[0059] Electric field sorting model

[0060] As mentioned above, the mechanical model of Dielectrophoresis shows that the force exerted by the electric field on the hair papilla cells is closely related to the cell size, dielectric constant and electric field gradient. Therefore, the most suitable electric field parameters can be calculated by optimizing the electric field intensity and frequency to maximize the cell separation efficiency.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for isolating and culturing autologous dermal papilla cells, characterized in that: The steps include: S1. Sample preparation: take skin tissue samples from the target individual, select the hair-dense area on the top of the head, use a scalpel and laser equipment to remove the hair follicles, use a micro blade and micro scissors to remove the skin tissue around the hair follicles, that is, the epidermis and part of the dermis, leaving only the hair follicle structure; S2. Microfluidic channel design. A microfluidic chip is designed. Multiple microchannels are set in the chip. The inner surface of the channel is coated with extracellular matrix molecules that bind to hair papilla cells, namely collagen and fibronectin, to enhance cell adhesion. The flow rate of the fluid is controlled so that the hair follicles are mechanically separated at a specific position when passing through the channel. The hair papilla cells are separated from other cells of the hair follicle through the pores of the microchannel. When the flow rate is v=10μm / s, the outer diameter of the hair follicle is 200-300μm, and the average diameter of the cells is 10μm-15μm, the hair papilla cells will preferentially attach to the chip surface. S3. Electric field sorting: introducing an alternating electric field or a direct electric field into the microfluidic channel, and by applying the principle of Dielectrophoresis and utilizing the polarization response of different cells to the electric field, the selective separation of hair papilla cells is achieved. The movement rate of hair papilla cells in the electric field is related to their dielectric constant. The dielectric constant of hair papilla cells will be repelled by the negative electric field. The electric field force formula is: Where r is the cell radius, m is the electric constant of the medium, K(ω) is the dielectric function of the cell, and E is the electric field strength. By adjusting the electric field strength and frequency, the hair papilla cells move to a specific position under the action of the electric field, thereby achieving their separation; S4, collecting and culturing, collecting the separated hair papilla cells into a preset culture dish or culture bottle through a microfluidic device, and using a customized culture medium containing growth factors, namely FGF and IGF, to promote the proliferation of the hair papilla cells; S5. Cell verification: The purity of the isolated cells was verified by immunofluorescence staining and Western blot detection of hair papilla cell-specific markers, namely CD34 and ALP. Anti-CD34 antibody was used for staining to observe whether there was a DPCs-specific fluorescence signal and to detect the expression of the hair papilla cell marker ALP.

2. The method for isolating and culturing autologous dermal papilla cells according to claim 1, characterized in that: The S3, electric field sorting, includes optimizing the relationship between electric field strength and frequency; Among them, low frequency enhances the cell aggregation effect by increasing the electric field strength; The high-frequency regulation of the electric field frequency causes "virtual separation" of cells, and the separation effect from other cells is maximized by adjusting the frequency.

3. The method for isolating and culturing autologous dermal papilla cells according to claim 2, characterized in that: The specific electric field formula is as follows: in, represents the frequency-dependent dielectric function, E is the electric field intensity, r is the cell radius, and m is the dielectric constant.

4. The method for isolating and culturing autologous dermal papilla cells according to claim 3, characterized in that: The low-frequency stage is optimized. Under low frequency, the hair papilla cells show positive DEP. At this time, the migration effect of the hair papilla cells is enhanced by increasing the electric field strength, and the electric field strength is set to 1kV / m-5kV / m.

5. The method for isolating and culturing autologous dermal papilla cells according to claim 4, characterized in that: The high frequency stage is optimized. At high frequency, the polarization effect of the cells is weakened. At this time, the electric field frequency is adjusted.

6. The method for isolating and culturing autologous dermal papilla cells according to claim 5, characterized in that: The S3, electric field sorting also includes frequency modulation and electric field waveform design. The electric field frequency is modulated into a variable frequency so that the hair papilla cells show different migration characteristics at different frequencies. Different waveforms such as sine waves, square waves or triangle waves are used. These waveforms will affect the response intensity and directionality of the cells to the electric field when adjusting the electric field frequency. By optimizing the waveform form, the sorting accuracy can be further improved.

7. The method for isolating and culturing autologous dermal papilla cells according to claim 6, characterized in that: The mathematical model between electric field strength and frequency is used for optimization analysis. When the electric field strength and frequency have a relationship with the cell migration speed, the following relationship is established: Among them, v cell is the migration speed of the cell, μ is the mobility of the cell in the electric field, E is the electric field strength, is the real part of the dielectric function.

8. The method for isolating and culturing autologous dermal papilla cells according to claim 7, characterized in that: The S3, electric field sorting, includes precisely controlling the electric field distribution, by precisely controlling the arrangement, spacing and shape of electrodes to achieve a more uniform and stable electric field distribution.

9. The method for isolating and culturing autologous dermal papilla cells according to claim 8, characterized in that: The S3 electric field sorting includes using multiple frequencies alternately, using low frequency and high frequency alternately, using frequency scanning technology, gradually increasing from low frequency to high frequency, mobilizing the negative and positive DEP characteristics of hair papilla cells, and optimizing the sorting process.

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