CN109136168B - Method for obtaining artificial hair papilla-like cells - Google Patents
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Abstract
The invention discloses a method for obtaining artificial hair papilla (DP) sample cells, which comprises the following steps: 1) preparing a mixed solution of sodium alginate and D-mannitol, and filtering and sterilizing; 2) extracting human dermal fibroblasts and culturing in vitro in large quantities; 3) obtaining sodium alginate microspheres wrapping human dermal fibroblasts by an electrostatic spraying method; 4) processing the sodium alginate microspheres to obtain APA microspheres; 5) APA microsphere is cultured in vitro to induce human dermal fibroblast to human hair papilla-like cells. According to the invention, sodium alginate microspheres are adopted to wrap human dermal fibroblasts to simulate the characteristic of DP cell aggregation and growth in a human body, and the method enables the human dermal fibroblasts to be transdifferentiated towards human hair papilla cells to obtain a large number of human hair papilla-like cells with hair generation induction capability, so that the problems that the human hair papilla cells are difficult to obtain and are easy to lose biological activity in vitro culture are effectively solved.
Description
Technical Field
The invention belongs to the field of biological materials and regenerative medicine, and particularly relates to a method for obtaining artificial hair papilla-like cells.
Background
Hair plays an important role in controlling body temperature, protecting skin, and the like, as an accessory organ of skin. Both diseases and medical treatments result in hair loss, which is not directly life-threatening to humans, but causes certain confusion to patients regarding psychology and quality of life. Currently, the most common cause of hair loss is androgenic alopecia, in which the early stage of hair follicle stem cells is not damaged, while the signal transduction of dermal hair papilla (DP) cells is disturbed, thereby failing to maintain hair growth. DP cells are therefore a critical target for the treatment of hair loss.
Currently, two methods, namely drug treatment and autologous hair follicle transplantation, are mainly adopted for treating alopecia. The drugs for treating alopecia obtained on the market have certain effects, but cannot promote the formation of new hair follicles, so that the treatment is temporary and permanent. Autologous hair follicle transplantation often requires a large amount of DP cells having a hair follicle-regenerating function. On one hand, the source of autologous human scalp hair papilla cells for transplantation in some severe alopecia patients is very limited, and on the other hand, the transplanted cells cannot be expanded, so that the feasibility of obtaining sufficient DP cells for surgical transplantation from patients clinically is not high, and a method for rapidly obtaining a large amount of DP cells with hair follicle regeneration function is urgently needed. The formation of hair follicles results from the interaction of the epidermis and dermis, where DP cells play a crucial role in inducing hair follicle regeneration and the hair cycle. DP cells lose their ability to induce hair regeneration rapidly when cultured in vitro, so how to obtain a large amount of DP cells while maintaining their ability to induce hair regeneration during in vitro culture has become a hotspot and difficulty in current research and development.
Research shows that the fibroblast has the multidirectional differentiation potential and can be differentiated into bone, cartilage, cardiac muscle, liver, nerve, pancreatic islet and other multiple embryonic layer tissues and organs under specific conditions. DP cells, as a specialized population of dermal fibroblasts, have many unique properties compared to other dermal fibroblasts, such as high alkaline phosphatase (ALP) activity and tendency to aggregate growth in vitro. Unlike the characteristics that DP cells are difficult to obtain and lose biological activity in vivo, fibroblasts are widely present in skin tissues, extraction and culture techniques are mature, the operation is simple, and physiological characteristics of DP cells are easy to maintain under in vitro culture conditions. The fibroblasts can be rapidly proliferated under the traditional culture condition, and if the fibroblasts can be effectively induced to be differentiated towards DP cells, a large amount of DP-like cells are hopefully obtained, so that a cell foundation is laid for autologous hair follicle transplantation.
Compared with the common plate culture, the three-dimensional environment can simulate the in vivo microenvironment better, and the biological material can provide a specific three-dimensional environment for cells in vitro. A large number of research reports show that cells can freely migrate in a sodium alginate hydrogel three-dimensional system prepared by an electrostatic spraying method, and a higher activity level can be maintained.
Patent 200410091871.5 discloses an artificial hair papilla substitute structure and its manufacturing method, wherein the hair papilla cells are coated with a hollow microcapsule of sodium alginate-polylysine-sodium alginate three-layer structure membrane, the diameter of the microcapsule is 200-. The artificial hair papilla substitute structure has the functions similar to those of hair papilla, has the functions of inducing hair follicle regeneration and maintaining hair growth, and can be prepared by artificial production in vitro. However, there are relatively few sources of papilla cells that can be clinically used for extraction, it is relatively difficult to extract rapidly growing papilla cells, stimulation by various growth factors needs to be added during extraction, and more importantly, the ability to induce hair growth is rapidly lost during in vitro culture. These above features greatly limit the clinical applications of the above patented methods.
In order to solve the problem of the DP cell source, researchers have attempted to induce dry cells, such as Embryonic Stem Cells (ESC), Induced Pluripotent Stem Cells (iPSC), Mesenchymal Stem Cells (MSC), and the like, to differentiate in the direction of hair papilla to become DP-like cells having the ability to promote hair follicle generation. However, the process is complicated, the induction cost is high, the efficiency of obtaining DP-like cells is low, and the stem cells have the risk of forming teratomas in vivo.
There have also been a great deal of research devoted to maintaining the function of DP cells in vitro, such as using growth factors and the like (wnt10b, PDGF and FGF2 and the like) to not only promote the proliferation of DP cells in vitro, but also maintain the ability of DP cells to induce hair production in vivo. However, a certain number of DP cells are required for DP function maintenance as a basis, and recombinant proteins are generally used to stimulate DP cells or transfect plasmids into DP cells, so that on one hand, the use of a large amount of recombinant proteins results in high economic cost, and on the other hand, the common transfection methods of two commonly used plasmids have unstable and low efficiency, and the difference between the transfection efficiencies of different batches is large.
There have also been many studies reporting that DP cells can be aggregated in vitro by methods such as the spinner method, suspension culture and the pendant drop method, and the DP-induced hair growth properties can be restored and hair follicle-like structures can be induced. However, similar to the technical solution disclosed in patent 200410091871.5, these studies also require obtaining a certain amount of DP cells with better induction ability, but actually clinical extraction of dermal papilla cells has relatively few sources, extraction of dermal papilla cells capable of rapidly growing is relatively difficult, and stimulation of various growth factors needs to be added during the extraction process, which is costly.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to develop an acquisition method of artificial human hair papilla-like cells. The method effectively solves the problems that DP cells are difficult to obtain and easy to lose biological activity in vitro, adopts the characteristic that the sodium alginate microspheres wrap human dermal fibroblasts to simulate the aggregation and growth of the DP cells in a human body, and ensures that the fibroblasts are subjected to transdifferentiation towards hair papilla cells by the simple and economic method, thereby finally obtaining a large amount of human hair papilla-like cells with the capacity of inducing hair generation.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for obtaining artificial hair papilla-like cells comprises the following steps:
1) extracting and culturing human dermal fibroblasts in vitro, and selecting human dermal fibroblasts within 10 generations;
2) respectively preparing 0.8-2% (g/ml) sodium alginate (sodium alginate) solution and 5-15% (g/ml) D-Mannitol (D-Mannitol) solution according to the mass-volume ratio, fully mixing and dissolving at room temperature, and then filtering and sterilizing to obtain sterile sodium alginate-Mannitol aqueous solution, which is hereinafter referred to as sodium alginate solution;
3) suspending human dermal fibroblast in 2) sodium alginate solution to obtain cell suspension containing at least 2 × 10 cells/ml6Personal dermal fibroblasts;
4) dispersing the cell suspension in the step 3) into micro-droplets by adopting an electrostatic spraying method, and dripping calcium chloride (CaCl) with the mass volume concentration of 1-2% (g/ml)2) Sodium alginate hydrogel microspheres which stably wrap human dermal fibroblasts are obtained from the solution; controlling the flow rate of the cell suspension propulsion pump to be 1-3ml/h, controlling the voltage of the electrostatic generating device to be 5-6kV, and controlling the duration of electrostatic spraying to be within 20 min;
5) sequentially treating the sodium alginate microspheres in the step 4) with 0.4-0.6(mg/ml) Polylysine (PLL) at 25-37 ℃ for 10-15min, treating with 0.08-0.2% (g/ml) sodium alginate solution for 4-10min, and treating with 5-6mM sodium citrate solution for 3-5min to finally obtain sodium alginate-polylysine-sodium alginate composite microspheres, hereinafter referred to as APA microspheres;
6) APA microspheres are cultured in a DMEM medium for 5-16 days, and human fibroblasts are gradually induced into human hair papilla-like cells.
Preferably, the step 2) is performed by filter sterilization by using a sterile filter with a pore size of 0.22 mu m and a sterile syringe.
Preferably, a mixed solution of 1% (g/ml) sodium alginate and 10% (g/ml) D-mannitol is prepared in the step 2) according to the mass-volume ratio.
Preferably, step 3) contains (2-10). times.10 per ml of suspension6Human dermal fibroblasts.
In a preferred scheme, the step 4) is specifically as follows: transferring the suspension into a 5ml sterile syringe, and replacing the syringe pillow with a 23-28G needle; the cell suspension in the step 3) is formed into micro-droplets with the diameter of about 350-2The human fibroblasts are distributed in the stable hydrogel microspheres in the solution to obtain solid microsphere precipitates.
Preferably, step 5) uses sterile normal saline to wash the obtained sodium alginate microspheres twice to remove residual CaCl in the spraying and receiving processes2Then adding DMEM culture solution containing 0.5mg/ml Polylysine (PLL) and treating at 25-37 deg.C for 10-15min to form a layer of PLL film on the microsphere surface.
Preferably, the PLL solution is removed in the step 5), 0.08-0.2% (g/ml) sodium alginate-0.5-1.5% (g/ml) D-mannitol solution is added for reaction for 4-10min, so that redundant PLL around the microsphere is removed, and a sodium alginate protective layer is formed outside the microsphere.
Preferably, the culture time of the human dermal fibroblasts in the APA microspheres in the step 6) is 8-10 days.
Preferably, in the step 6), DMEM high-sugar medium containing 10-20% by volume of fetal calf serum and 1% by volume of penicillin/streptomycin is used for culturing the microsphere-embedded cells, and the medium is replaced every two days.
The invention will be further explained and illustrated below:
the present inventors have found that dermal fibroblast precursor cells can induce differentiation into dermal fibroblasts or dermal aggregates, dermal hair papilla cells, dermal sheath cells, etc., which function as matrix secretion in developing hair follicles, indicating that human dermal fibroblasts and hair papilla (DP) cells have homology. On the other hand, DP cells, as a specialized population of dermal fibroblasts, have many unique properties compared to other dermal fibroblasts, such as high alkaline phosphatase (ALP) activity and tendency to aggregate in vitro. Unlike the characteristics that DP cells are difficult to obtain and lose biological activity in vivo, fibroblasts are widely present in skin tissues, extraction and culture techniques are mature, the operation is simple, and physiological characteristics of DP cells are easy to maintain under in vitro culture conditions. The fibroblasts can be rapidly proliferated under the traditional culture condition, and if the fibroblasts can be effectively induced to be differentiated towards DP cells, a large amount of DP-like cells are hopefully obtained, so that a cell foundation is laid for autologous hair follicle transplantation. The invention adopts the characteristic that the sodium alginate microspheres wrap the human dermal fibroblast to simulate the growth of DP cells aggregated in organisms, and the fibroblast is subjected to transdifferentiation towards the direction of hair papilla cells by the simple and economic method, so that a large amount of hair papilla-like cells with hair generating capacity are finally obtained.
The main object of the present invention is to provide an artificial hair papilla-like cell which can rapidly obtain a large amount of hair papilla cells having characteristics of hair papilla cells, i.e., inducing hair growth, in vitro. The human dermal fibroblast is simply wrapped by sodium alginate hydrogel to form hydrogel microspheres, and the hydrogel microspheres are cultured for a certain time, so that the human dermal fibroblast has the capacity of inducing hair growth. The invention relates to three aspects, including human dermal fibroblast, a sodium alginate-based cell wrapping material and a microcapsule preparation technology for realizing cell wrapping. Wherein, the human dermal fibroblast is easy to obtain and can be cultured in a large scale under simple conditions, thereby laying a cellular foundation for the later induction. In fact, the dermal fibroblast fluid may be replaced with other dermal fibroblasts, such as mouse, rat, rabbit, etc. In a second aspect, the sodium alginate material used to encapsulate the seed cells is widely available and readily available. In a third aspect, the shape of the sodium alginate microspheres wrapping human dermal fibroblasts obtained by the electrostatic spraying method is basically similar to that of a human papilla basement membrane.
The method for obtaining the hair papilla-like cells sequentially comprises the following main steps: 1) preparing a mixed solution of sodium alginate and D-mannitol with the mass volume ratio of 1%, and filtering and sterilizing; 2) extracting human dermal fibroblasts and culturing in vitro in large quantities; 3) obtaining sodium alginate microspheres wrapping human dermal fibroblasts by an electrostatic spraying method; 4) respectively treating the sodium alginate microspheres with polylysine and sodium citrate solution to obtain APA microspheres; 5) after APA microspheres are cultured in vitro for a period of time, human fibroblasts are induced into human hair papilla-like cells. The obtained human hair papilla-like cells and epidermal cells of a C57 newborn mouse are co-injected into the subcutaneous back skin of a nude mouse, the growth condition of hair of the injected part is observed after four weeks, and the C57 epidermal cells and dermal cells of the newborn mouse are co-injected into the subcutaneous back skin of the nude mouse as a positive control.
The steps are further described in detail:
the selection of the state and the generation number of the human dermal fibroblasts themselves in step 1) is important. Generally, the better the state of human dermal fibroblasts, the stronger the induction ability of hair after three-dimensional culture by APA microspheres. Further, it is preferable to select human dermal fibroblasts within 10 generations for induction, and after 10 generations, the larger the generation number, the weaker the hair induction ability, to about 13 generations, and it is finally difficult to induce hair generation in vivo even in the induction environment of APA microspheres.
The mass-volume ratio of mannitol in the step 2) has no great influence on the formation of microspheres by the mixed solution, and the mass-volume ratio of sodium alginate in a certain range is larger, the viscosity of the mixed solution is larger, and the too large viscosity can be unfavorable for the migration of human dermal fibroblasts in the microspheres; the lower the concentration of sodium alginate, the more efficient the human dermal fibroblasts can move in an embedded state to achieve sufficient cell-cell and cell-matrix contact, thereby forming the ideal human dermal fibroblast aggregation similar to DP aggregation.
Step 3) is that human dermis becomes fiber fineThe key to the success of cell induction to human hair papilla-like cells. The following points should be mainly noted: first, the state of human dermal fibroblasts themselves greatly affects the success rate of induction. Second, the generation number of the selected human dermal fibroblasts is about 10 th generation at the latest. Thirdly, the packing density of the cells in the sodium alginate cannot be too low, and at least 2X 10 should be ensured6One per ml. In addition, the electrostatic spraying time is not suitable to be too long, and is controlled to be completed within 20min as much as possible, so that the influence of the preparation process on the cell activity is reduced as much as possible.
And 4) controlling the flow rate of the cell suspension propulsion pump to be 1-3ml/h, wherein the diameter of the microspheres is not obviously changed along with the increase of the propulsion speed of the pump, but the overall duration of electrostatic spraying is directly influenced. In contrast, the faster the flow rate of the boost pump, the shorter the electrostatic spray time.
And 4) controlling the voltage of the static electricity generating device to be 5-6kV, wherein the diameter of the sodium alginate microspheres obtained in the range is reduced along with the increase of the voltage. When the voltage exceeds 6kV, the microspheres do not present standard spherical shapes, but present trailing tail-shaped structures, and the tail-shaped structures are more and more obvious along with further increase of the voltage, so that most microspheres no longer present complete spherical appearances. Under the condition, cells in the microspheres may be unevenly distributed, and meanwhile, external nutrient substances and cell metabolic wastes may be deposited at a certain position in the microspheres, so that effective substance exchange and information exchange cannot be performed, and further, the activity and the induction efficiency of the cells are adversely affected. When the voltage between the generating device and the receiving device is 5kV, the diameter of the obtained microsphere is about 550 μm; whereas the diameter of the microspheres is about 350 μm when the voltage between the generating means and the receiving means is 6 kV.
And 4) controlling the duration of electrostatic spraying within 20 min. Throughout the spraying process, human dermal fibroblasts will be left at room temperature and nutrient and gas exchange will be limited. Under the condition, the cell activity can be damaged by the long spraying time, and the subsequent experiment results are influenced.
Step 5) washing the sodium alginate microspheres obtained by the step by using sterile normal salineTwice to remove residual CaCl during spraying and receiving2Then adding DMEM culture solution containing PLL of 0.5mg/ml to treat at 25-37 deg.C for 10-15min to form a layer of PLL film on the microsphere surface.
Culturing the human dermal fibroblasts in the APA microspheres for 5-16d in the step 6), wherein the human dermal fibroblasts can detect the increase of the DP specific marker gene in 5-16 days, but the highest point of the increase is about 9 days after the APA microspheres are cultured, and then the increase is reduced.
The human dermal fibroblasts cultured in the APA microspheres are a mixed cell group and comprise artificial human dermal papilla-like cells, human dermal fibroblasts, neural crest cells and the like, but the percentage content of the artificial human dermal papilla-like cells is gradually increased along with the increase of the culture time within a certain time range. The general rule is that the highest percentage of human dermal fibroblasts are induced to be human hair papilla-like cells after the human dermal fibroblasts are cultured in the APA microspheres for about 9-10 days, and then the percentage of the human hair papilla-like cells is gradually reduced.
The room temperature of the invention is about 25 ℃ and between 20 ℃ and 30 ℃.
Compared with the prior art, the invention has the technical advantages that:
1. the whole operation process is simple, sodium alginate is easy to obtain as a raw material, and the method is economical and cheap, and does not need to use various recombinant proteins and stimulating factors for culture and induction;
2. compared with the common plate culture, the APA microsphere culture can better simulate the characteristic of DP aggregation growth in vivo;
3. human dermal fibroblasts are easily obtained and are easy to culture on a large scale;
4. the human dermal fibroblast is derived from autologous cells, so that immunological rejection can be avoided;
5. human dermal fibroblasts can obtain the capacity of inducing hair regeneration in a short time under the condition of APA microsphere culture.
Drawings
FIG. 1 is a process flow diagram of the present invention. (1) The preparation contains 1 percent of sodium alginate and 10 percent of sodium alginateMixed solution of mannitol. (2) Human dermal fibroblasts were extracted and cultured in vitro in large quantities. (3) The cell density in the mixture was 6X 106And when the sodium alginate microspheres are used for per ml, the sodium alginate microspheres are obtained by an electrostatic spraying method, wherein the voltage is about 5.5 KV. (4) And sequentially carrying out PLL (phase locked loop) wrapping, 0.1% sodium alginate solution treatment and 5.5mM sodium citrate solution treatment on the obtained sodium alginate microspheres to obtain the APA microspheres. (5) Epidermal cells of newborn mice and APA-coated dermal cells of the mice were injected into the backs of the nude mice, and hair growth was observed after 4 weeks.
FIG. 2 is a graph of data from an experiment in which human dermal fibroblasts encapsulated with APA microspheres were induced into human dermal papilla-like cells. A: growth of human dermal fibroblasts in APA microspheres. B: human dermal fibroblasts were cultured in APA microspheres for 1d, 5d and 9d alkaline phosphatase (ALP) activity profiles. C: human dermal fibroblasts were cultured in APA microspheres for 1d, 5d and 9d as a-SMA positive cells. As shown in fig. a, the human dermal fibroblasts that have just been encapsulated are uniformly dispersed in the APA microspheres. B shows that the activity of alkaline phosphatase gradually increases with the increase of the culture time of the APA microspheres, and the ALP activity after 1d, 5d and 9d culture is 6.05, 8.92 and 10.16 King units/gprot, respectively. In the C picture, the percentage of 1d, 5d and 9d of the flow detection a-SMA positive cells after culture is 8.83%, 13.8% and 17.5%, respectively. It can be seen that the increase of the culture time of the human dermal fibroblasts in the APA microspheres shows that the protein expression levels of the two human dermal papilla specific marker genes (ALP and a-SMA) gradually increase in the culture time of 1d, 5d and 9d, further suggesting that the human dermal fibroblasts can be partially transdifferentiated into human dermal papilla-like cells under the three-dimensional culture condition of the APA microspheres.
FIG. 3 is a graph of the induction of hair regeneration in vivo by human dermal fibroblasts encapsulated with APA microspheres. A: nude mice were injected subcutaneously on their backs with epidermal cells from C57 neonatal mice. B: the dorsal part of the nude mice was injected subcutaneously with epidermal cells and dermal cells of C57 neonatal mice. C: subcutaneous C57 neonatal murine epidermal cells on the back of nude mice and human dermal fibroblasts cultured in plate for 9 days. D and E: the dorsal part of the nude mouse is injected with C57 newborn mouse epidermal cells and APA microspheres to wrap the human dermal fibroblasts for 9 days. Hair generation requires interaction of epidermis and mesenchyme, and simple C57 neonatal murine epidermal cells as shown in panel a are not sufficient to induce hair regeneration, as a negative control. C57 neonatal murine epidermal cells and C57 neonatal dermal cells induced hair regrowth, as shown by the generation of hair follicle structures in panel B, as positive controls. The neonatal murine dermal cells used in panel B contain, in addition to fibroblast progenitor cells, other components that stimulate hair growth and development, such as microvessels, dermal extracellular matrix, and melanin progenitor cells, and neonatal murine dermal cells over 2 days do not have the ability to induce hair growth. The apparent melanin pigment deposition and the newly induced hair-like structures such as hair shafts can be seen in panels D and E, where the arrows indicate the newly formed hair shafts. It is suggested that human dermal fibroblasts encapsulated under three-dimensional culture conditions of APA microspheres can induce hair generation. Since nude mice have BALB/C (albino) genetic background and produce white hair, and the color of the hair depends on epidermal cells, a black newborn mouse with C57 genetic background was selected to distinguish whether newly formed hair is nude mice themselves or induced. On the other hand, the generation of black hair-like structures is hardly seen in panel a, indicating that the C57 neonatal murine epidermal cells used for the experiment are not contaminated with C57 neonatal murine dermal cells, further indicating that human dermal fibroblasts are able to induce DP-like cells under three-dimensional conditions. The graph C is similar to the graph A, and no hair-like structure is generated, namely the human dermal fibroblasts cultured by the common plate do not have the hair induction capability, and the importance of the three-dimensional environment cultured by the APA microspheres in the induction of the human dermal fibroblasts to the human hair-like cells is further proved.
Fig. 4 is a graph of the generation of newly formed hair-like structures derived from the induction of human dermal fibroblasts in APA microspheres. As shown in the figure, green fluorescence is K14 labeled epidermal cells, and red fluorescence is a human specific mitochondrial marker gene. It is suggested that the production of hair-like structures is at least partially due to the production of human dermal fibroblasts following induction.
Detailed Description
The invention will be further explained and explained with reference to the drawings and the embodiments
Example 1
As shown in FIG. 1, the method for obtaining the artificial hair papilla-like cells sequentially comprises the following main steps:
1) preparing a mixed solution of 1% sodium alginate and 10% D-mannitol according to the mass-volume ratio, fully mixing, dissolving overnight at room temperature, and filtering and sterilizing the hydrogel in a biological safety cabinet by using a sterilizing filter with a pore size of 0.22 mu m and a sterile syringe after fully dissolving to obtain sterile sodium alginate hydrogel;
2) extracting human dermal fibroblasts and culturing in vitro in large quantities;
3) suspending human dermal fibroblast in 2) sodium alginate solution to obtain suspension containing 6 × 10/ml6Personal dermal fibroblasts;
4) transferring the hydrogel suspension mixed with human dermal fibroblasts with proper concentration into a 5ml sterile syringe, and replacing the syringe pillow with a needle with a 23G point;
5) the cell suspension in the 4) is formed into a micro-droplet state with the diameter of about 350-2In the solution, the fibroblasts are positioned in the stable hydrogel microspheres to obtain solid microsphere precipitates; electrostatic spraying parameters of the obtained microspheres: the flow rate of the electrostatic propulsion pump is 3ml/h, the voltage of the electrostatic generating device is 5-6kV, and the electrostatic spraying time is not longer than 20 min.
6) Washing the obtained sodium alginate microspheres twice by using sterile normal saline to remove a large amount of CaCl in a receiving device in the spraying process2Then adding DMEM culture solution containing 0.5mg/ml PLL at 37 ℃ for 10min to enable the PLL to form a layer of film on the surface of the microsphere and help to fix the form of the microsphere;
7) removing the PLL solution, adding 0.1% sodium alginate-1% mannitol solution, reacting for 4min to remove the surplus PLL around the microsphere, and forming a loose sodium alginate protective layer outside the microsphere. After the reaction was completed, 5.5mM sodium citrate solution was added for 3min to remove the residual Ca2+And slightly dissolve sodium alginate and Ca2+Formed ofAnd the membrane makes the membrane structure on the periphery of the microsphere looser. At the moment, the protective film of the PLL can support the microsphere to maintain the spherical shape of the microsphere, and after the PLL is properly dissolved, the internal space structure of the sodium alginate microsphere becomes looser, so that the movement resistance of cells in the microsphere is reduced, and the discharge of cell metabolic waste and the entry of external nutrient substances are facilitated. After the reaction is finished, the microspheres are washed by a small amount of DMEM (DMEM), then a proper amount of culture solution is added for culture, the DMEM high-glucose culture medium containing 20% fetal calf serum, 1% streptomycin and 1 Xglutamine mixed solution is used for culturing the cells embedded in the microspheres, and the culture medium is replaced every two days.
And (3) verification: the alkaline phosphatase activity and the percentage of a-SMA positive cells of human dermal fibroblasts after being cultured in the APA microspheres for 9 days are remarkably increased compared with that of the human dermal fibroblasts cultured for 1d (figure 2), and the increase of the protein levels of the alkaline phosphatase and the a-SMA as two DP specific marker genes suggests that the APA microspheres are used for wrapping to induce the human dermal fibroblasts to be transformed into hair papilla-like cells. After the hair-like cells having a hair-inducing function were obtained as described above, the hair-like cells and epidermal cells of newborn mice were co-injected into the backs of nude mice to reconstitute hair follicles.
The main technical routes involved in hair follicle reconstruction include:
1) respectively obtaining epidermal cells and dermal cells of a C57 newborn mouse;
2) skin reconstruction surgery: mixing the hair papilla-like cells induced by sodium alginate and C57 newborn mouse epidermal cells in a ratio of 2:1, and injecting the mixed cells into the back of a nude mouse by a subcutaneous injection method.
3) And (3) morphological observation: after four weeks, the injection site was observed for hair growth.
The test results are as follows: four weeks after transplantation, the formation of a number of similar hair follicle structures was seen in the area of dorsal subcutaneous tissue injection (fig. 3). The K14-labeled epidermal cells and the mitochondrion-labeled human-derived cells further prove that the generated hair follicle-like structures are generated by being induced by human dermal fibroblasts (figure 4), and the hair follicle-like structures are formed by arranging concentric circular structures of epithelial cells, and the test results show that the APA microcapsules can induce partial conversion of the human dermal fibroblasts into hair papilla-like cells capable of inducing hair follicle regeneration and maintaining hair growth.
Claims (1)
1. A method for obtaining artificial hair papilla-like cells comprises the following steps:
1) extracting and culturing human dermal fibroblasts in vitro, and selecting human dermal fibroblasts within 10 generations; preparing a mixed solution of 1% sodium alginate and 10% D-mannitol according to the mass-volume ratio, fully mixing, dissolving overnight at room temperature, and performing filtration sterilization in a biological safety cabinet by using a sterilization filter with a pore size of 0.22 mu m and a sterile syringe after fully dissolving to obtain sterile sodium alginate hydrogel;
2) extracting human dermal fibroblasts and culturing in vitro in large quantities;
3) suspending human dermal fibroblast in 1) prepared sodium alginate hydrogel to form cell suspension containing 6 × 10 cells per ml6Personal dermal fibroblasts;
4) transferring the cell suspension liquid in the step (3) into a 5ml sterile syringe, and replacing the needle of the syringe with a 23G needle;
5) the cell suspension in the step 4) is formed into a droplet state with the diameter of 350-550 mu m by using a high-voltage electric field microcapsule generator, and then CaCl with the concentration of 2 percent is dripped2In the solution, the fibroblasts are positioned in the stable hydrogel microspheres to obtain solid microsphere precipitates; electrostatic spraying parameters of the obtained microspheres: the flow rate of the electrostatic propulsion pump is 3ml/h, the voltage of the electrostatic generating device is 5-6kV, and the electrostatic spraying time is not longer than 20 min;
6) washing the microspheres obtained in the step (5) twice by using sterile normal saline to remove a large amount of CaCl in a receiving device in the spraying process2Then adding DMEM culture solution containing 0.5mg/ml PLL at 37 ℃ for 10min to enable the PLL to form a layer of film on the surface of the microsphere and help to fix the form of the microsphere;
7) removing PLL solution, adding 0.1% sodium alginate-1% mannitol solution, and reacting for 4min to removeRedundant PLL around the microsphere and a loose sodium alginate protective layer is formed outside the microsphere; after the reaction was completed, 5.5mM sodium citrate solution was added for 3min to remove the residual Ca2+ And slightly dissolve sodium alginate and Ca2+ The formed film; after the reaction is finished, the microspheres are washed by DMEM, then culture solution is added for culture, DMEM high-sugar culture medium containing 20% fetal calf serum, 1% streptomycin and 1 Xglutamine mixed solution is used for culturing the cells embedded in the microspheres, and the culture medium is replaced every two days for 9 days.
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| CN1650823A (en) * | 2004-12-24 | 2005-08-10 | 李宇 | Artificial hair mammilla substition structure and its manufacturing method |
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