Multilayered tissue engineered skin consisting of hair follicle cells, methods of preparation and uses thereof

Abstract

The present invention relates to a multi-layered tissue engineered skin made of hair follicle cells, its preparation method and use. The preparation method includes digestion of autologous hair follicles in vitro, expansion culture of hair follicle mixed cells, maturation differentiation of hair follicle mixed cells, and formation of tissue structure. Compared with the prior art, the tissue engineered skin according to the present invention is composed only of autologous hair follicle cells, forms a layered structure such as physiological basal layer, spinous layer and granular layer, and contains abundant melanocytes in the basal layer. The tissue engineered skin has tight intercellular junctions and good toughness, and contains abundant basal layer-like cells that actively divide, and the abundant melanocytes promote the re-pigmentation of skin in vitiligo areas, widening the field of use of the tissue engineered skin. The present invention uses autologous hair follicles derived from a wide range of sources, reducing the industrialization cost.

Description

[Technical Field]

[0001] The present invention relates to the field of biology, in particular to multi-layered tissue engineered skin consisting of hair follicle cells, its production method and use. [Background technology]

[0002] Skin is one of the largest organs in the human body, covering the entire body surface and accounting for approximately 16% of body weight. It has important functions such as secretion, absorption, body protection, sensation, and immunity. Physiological skin is divided into the epidermis, dermis, and subcutaneous tissue. Trauma, burns, inflammation, and ulcers often cause loss of all layers of skin. Autoimmune diseases such as vitiligo can also cause a loss of melanocytes in the epidermis, resulting in abnormal skin color. Tissue-engineered skin is the earliest, fastest-growing, and most technologically mature tissue-engineered organ. Numerous experimental and clinical studies have demonstrated that tissue-engineered skin can be used as a substitute for skin wound repair and reconstruction, and mature tissue-engineered skin products have already been launched both in China and abroad. In the field of vitiligo treatment, tissue-engineered epidermal products have yet to appear, primarily due to the difficulty of developing a mixed skin cell culture system free of exogenous components. Furthermore, during conventional in vitro culture, epidermal cells tend to lose their keratinization characteristics, which increases the fragility of the resulting tissue-engineered epidermal skin fragments and makes it difficult for them to form structural characteristics similar to those of physiological epidermis, thereby affecting their function.

[0003] Cell activity is particularly important in tissue-engineered skin. If the majority of cells are already differentiated, stem cells are scarce, and the repair wound is large, the differentiated cells will gradually senesce and fall off. Without sufficient stem cells to replenish them or cells that migrate to the surrounding area, the graft will not survive. Therefore, sufficient stem cells are crucial for tissue-engineered skin and are crucial for ensuring cell renewal and long-term survival of the graft in the wound. Hair follicle epithelial cells are structurally continuous with keratinocytes in the epidermal layer, share similar cellular morphology, and can transform into each other under certain conditions. Hair follicle epithelial cells are primarily composed of outer root sheath cells of hair matrix cells. By analyzing the clonogenic potential of hair follicle cells from different parts of the skin, we discovered cells with high proliferative potential in the hair follicle bulge region, demonstrating the presence of stem cells in the hair follicle bulge region. In recent years, the hair follicle bulge region has been recognized as a reservoir of stem cells. They not only participate in hair follicle formation and maintain cyclical hair growth, but also play an important role in epidermal development, forming transient amplifying cells in the basal layer of the epidermis, maintaining epidermal self-renewal, and repairing the epidermis during skin injury. Melanocyte stem cells have also been found in the hair follicle bulge region. Therefore, hair follicles are a superior source of seed cells for tissue-engineered skin. Research has shown that skin flakes derived from hair follicle outer root sheath cells have stronger adhesive properties, forming an "edge effect," and that the flake cells release large amounts of growth factors and cytokines, stimulating epithelial cell migration and promoting wound contraction and wound size reduction. This phenomenon has not been reported with conventional skin epidermal grafts or conventional monolayer-cultured keratinocyte grafts.

[0004] Existing cell culture systems for tissue-engineered skin products using hair follicle epithelial cells as seed cells and containing live cells rely heavily on serum and fibroblast trophoblasts. The use of culture reagents, culture conditions, serum, types of exogenous factors, and cell culture density can lead to differences in cell heterogeneity, differentiation potential, proliferation potential, and cell function after expansion culture, limiting the standardized production and medical use of skin fragments. Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to provide a multi-layered tissue engineered skin consisting of hair follicle cells, a method for producing same and use thereof, in order to overcome the above-mentioned deficiencies existing in the prior art.

[0006] The multilayered tissue-engineered skin of the present invention is composed only of autologous hair follicle cells and forms a layered structure of the basal layer, spinous layer, and granular layer, which are rich in melanocytes, just like physiological skin. [Means for solving the problem]

[0007] The object of the present invention can be achieved by the following technical solutions.

[0008] The present invention provides a method for producing multi-layered tissue-engineered skin composed of hair follicle cells, comprising: The method includes the steps of digesting autologous hair follicles in vitro, expanding and culturing the mixed hair follicle cells, and maturing and differentiating the mixed hair follicle cells and forming a tissue structure, In the step of digesting the autologous hair follicles in vitro, The hair follicle tissue is digested by a two-stage enzymatic digestion method, and the hair follicle bulge region, which mainly contains hair follicle stem cells and melanocytes, the outer root sheath, which is rich in keratinocytes, and the hair follicle region, which is rich in dermal papilla cells, are effectively digested to obtain mixed hair follicle cells; In the step of expanding and culturing the hair follicle mixed cells, The single cell suspension containing mixed hair follicle cells was expanded in two stages using culture medium A and culture medium B to obtain mixed hair follicle cells after expansion culture. In the step of maturation and differentiation of the hair follicle mixed cells and formation of a tissue structure, After expanding and culturing the hair follicle mixed cells in culture medium B until they reach a confluence rate of 80%, they are then cultured in culture medium C. The culture medium is changed daily during culture in culture medium C, and the hair follicle mixed cells, which are rich in hair follicle stem cells, pre-melanocytes, and keratinocytes, are induced to differentiate into mature epidermal cells in culture medium C. The mature cells are arranged in layers, and the epidermal cells are tightly bound together by the action of culture medium C, forming multi-layered tissue-engineered skin composed of hair follicle cells. The culture solution A is When the volume is 500 ml, the ingredients are 500 ml of a commercially available keratinocyte medium with clearly defined ingredients, insulin-like growth factor 1-10 ng / ml, keratinocyte growth factor 0.5-5 ng / ml, endothelin 100-500 ng / ml, melanocyte-stimulating hormone α 20-80 ng / ml, and gentamicin 20-100 μg / ml. The culture medium B is When the volume is 500 ml, the ingredients are 500 ml of a commercially available keratinocyte medium with clearly defined ingredients, insulin-like growth factor 1-10 ng / ml, basic fibroblast growth factor 1-10 ng / ml, epidermal growth factor 10-50 ng / ml, adrenocorticotropic hormone 10-50 ng / ml, and melanocyte-stimulating hormone α 20-80 ng / ml, The culture solution C is The present invention provides a production method in which, when the volume is adjusted to 500 ml, the ingredients are 370 ml of commercially available DMEM medium, 125 ml of commercially available F12 medium, 5 ml of non-essential amino acids, 1 to 10 ng / ml of insulin-like growth factor, 1 to 10 μg / ml of taurine, 1 to 5 μg / ml of 5-hydroxytryptamine hydrochloride, 20 to 80 μg / ml of insulin, 10 to 50 μg / ml of recombinant transferrin, 1 to 10 ng / ml of basic fibroblast growth factor, 1 to 10 nM of triiodothyronine, 0.5 to 5 μg / ml of hydrocortisone, 0.5 to 5 ng / ml of keratinocyte growth factor, 10 to 50 ng / ml of epidermal growth factor, 10 to 50 ng / ml of adrenocorticotropic hormone, and 10 to 50 μg / ml of adenosine.

[0009] In one embodiment of the present invention, the ex vivo autologous hair follicles are hair follicles isolated from the human body, i.e. the method according to the present application is carried out on ex vivo tissue.

[0010] In one embodiment of the present invention, a conventional technique may be used to harvest extracorporeal autologous hair follicles, such as by extracting hair follicles using a hair follicle remover. A specific extraction method may be the FUE (follicle unit extraction) method used in hair transplantation. For example, see "Follicular Unit Extraction: Minimally Invasive Surgery for Hair Transplantation." Dermatol Surg. 2002; 28: 720-7. The tip of the hair follicle harvester is pierced into the scalp skin under an electric press, and the hair follicles are then extracted gently with sterile elbow tweezers, with the structure of the hair follicles being completely preserved throughout the entire process.

[0011] In one embodiment of the present invention, a method for digesting hair follicle tissue by a two-step enzymatic digestion method includes a first enzymatic digestion and a second enzymatic digestion, In the first enzymatic digestion, the in vitro autologous hair follicle tissue was immersed in culture solution A containing 0.6 to 2.4 U / ml of neutral protease and incubated overnight at 37°C. For the second enzymatic digestion, the next day, the hair follicle tissue is minced, TrypLE is added, and digestion is carried out at 37°C for 10 to 40 minutes, followed by vigorously pipetting. After pipetting, the suspension containing the cells and tissue is filtered through a 100 μm sieve, and the filtrate is centrifuged. After that, phosphate buffer is added to the cell mass, and the mixture is washed 3 to 5 times. Culture medium A is then added to produce a single-cell suspension of hair follicle cells containing mixed hair follicle cells.

[0012] In one embodiment of the present invention, in a two-stage expansion culture method of a hair follicle single cell suspension containing mixed hair follicle cells using culture medium A and culture medium B, 10 mixed hair follicle cells 4 ~10 6 The cells were inoculated at a cell concentration of / ml onto a culture dish coated with a substrate for 4 hours, and culture medium A was added and expanded under conditions of 5% CO2 and 37°C. The liquid was changed every other day, and the cells were passaged when the confluence rate reached 80% or more. After passage, the cells were 4 ~10 6The cells are seeded at a cell concentration of / ml onto a culture dish coated with a substrate for 4 hours, and culture medium B is added. The cells are expanded at 5% CO2 and 37°C. The liquid is changed every other day. When the simultaneous expansion of hair follicle stem cells, pre-melanocytes, and keratinocytes is completed, mixed hair follicle cells are obtained after expansion.

[0013] In one embodiment of the present invention, the substrate for covering a culture dish comprises, as its components, 500 ml of commercially available phosphate buffer solution and 10 to 100 μg / ml of type IV collagen when the volume is adjusted to 500 ml.

[0014] In one embodiment of the present invention, in the maturation differentiation of hair follicle mixed cells and formation of tissue structures, the hair follicle mixed cells after expansion culture are cultured for 10 4 ~10 5 pieces / cm 2 The cells are inoculated onto a substrate-coated culture dish at a density of 10 ...

[0015] The present invention further provides multi-layered tissue engineered skin produced by the above-described production method.

[0016] In one embodiment of the present invention, the multi-layered tissue engineered skin comprises melanoblasts and stem keratinocytes resembling physiological conditions.

[0017] In one embodiment of the present invention, the multilayered tissue-engineered skin is a stratified tissue structure having a physiological basal layer, a spinous layer, and a granular layer, and the basal layer is rich in melanoblasts. The tissue-engineered skin is rich in actively dividing basal layer-like cells with tight intercellular junctions and good toughness.

[0018] The present invention further provides a use of multi-layered tissue-engineered skin produced by the above-described method, wherein the multi-layered tissue-engineered skin is used for the production of biomedical materials, and is useful for the treatment of pigmentation defects and epidermal deficiency disorders.

[0019] In one embodiment of the present invention, the fabricated multilayered tissue engineered skin, when used for stable vitiligo treatment, can promote repigmentation of vitiligo and achieve healing of vitiligo without immune rejection due to its autologous origin. [Effects of the Invention]

[0020] The method for producing multilayered tissue-engineered skin composed of hair follicle cells according to the present invention involves the in vitro digestion of autologous hair follicles, the expansion and culture of mixed hair follicle cells, the maturation and differentiation of the mixed hair follicle cells, and the formation of a tissue structure. The production method according to the present invention does not contain any exogenous components in the culture system, and the tissue-engineered epidermis produced is composed only of autologous cells, without the presence of allogeneic or xenotrophoblastic cells, making the product safer for clinical use. The cellular composition is a mixture of cells, primarily melanocytes and keratinocytes. The keratinocytes form a multilayered structure, with a large number of melanocytes arranged above a basal layer similar to that of physiological epidermis. Due to the properties of these cells, the tissue-engineered epidermis can be used for a wide range of indications, including surgical transplantation treatment of stable vitiligo, and can promote repigmentation of vitiligo.

[0021] The present invention uses autologous hair follicles as a seed cell source for tissue-engineered skin, significantly reducing material-derived limitations and donor pain. The autologous cells avoid immune rejection after transplantation, favoring their integration into the tissue-engineered skin and the abrasion wound surface, achieving better therapeutic effects. The hair follicles used contain abundant stem cells, have active cell metabolism, high proliferation potential, and have a high number of passages, which is favorable for large-scale expansion of seed cells, reducing the amount of material harvested and industrialization costs.

[0022] The present invention involves harvesting autologous hair follicles, isolating hair follicle epithelial keratinocytes, premelanocytes, and hair follicle stem cells through enzymatic digestion, efficiently amplifying and culturing them in vitro, and then inducing their differentiation into mature epidermal cells. After culturing, the resulting tissue-engineered skin is composed solely of autologous hair follicle cells, forming a layered structure resembling the physiological basal, spinous, and granular layers, with abundant melanocytes in the basal layer, resulting in a multilayered tissue structure similar to the physiological state. This tissue-engineered skin possesses tight intercellular junctions, good toughness, and an abundance of actively dividing basal layer-like cells, increasing the success rate of transplantation. The abundant melanocytes promote skin repigmentation in vitiligo areas, thereby expanding the scope of use of tissue-engineered skin. Because autologous hair follicles are harvested, the harvesting process is easy to obtain, painless, and reduces industrialization costs.

[0023] The research of the present invention has confirmed that it is biotechnologically feasible to construct a culture system of hair follicle-derived mixed cells with defined components and free of serum and nutrient layers, and that hair follicle-derived mixed cells can be used as seed cells for tissue-engineered epidermis to treat vitiligo. [Brief explanation of the drawings]

[0024] [Figure 1] 1 shows the cell morphology of mixed hair follicle cells in culture medium A in Example 1 (10x objective lens). [Figure 2] 1 shows the cell morphology of mixed hair follicle cells in culture medium B in Example 1 (10x objective lens). [Figure 3] 1 shows the cell morphology of mixed hair follicle cells in culture medium C in Example 1 (10x objective lens). [Figure 4] 1 shows the morphology of melanocytes in hair follicle mixed cells in Example 1 (10x objective lens). [Figure 5] 1 shows the multi-layered tissue-engineered skin (area: 21 cm 2 ) made of hair follicle cells in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0025] The invention will now be described in detail with reference to the drawings and specific embodiments.

[0026] In the following examples, experimental methods for which no specific conditions are specified generally follow conventional conditions or conditions suggested by the manufacturer.

[0027] Example 1 Step 1: Prepare the culture medium Preparation of coated substrate: When the volume is 500 ml, the components are 500 ml of commercially available phosphate buffer solution and 30 μg / ml of type IV collagen. Preparation of culture medium A: When the volume is 500 ml, the ingredients are 500 ml of a commercially available keratinocyte medium with clearly defined components, insulin-like growth factor 10 ng / ml, keratinocyte growth factor 3 ng / ml, endothelin 200 ng / ml, melanocyte-stimulating hormone α 50 ng / ml, and gentamicin 50 μg / ml. Preparation of culture medium B: When the volume is 500 ml, the ingredients are 500 ml of a commercially available keratinocyte medium with clearly defined components, insulin-like growth factor 10 ng / ml, basic fibroblast growth factor 4 ng / ml, epidermal growth factor 20 ng / ml, adrenocorticotropic hormone 50 ng / ml, and melanocyte-stimulating hormone α 50 ng / ml. Preparation of culture medium C: When the volume is adjusted to 500 ml, the ingredients are 370 ml of commercially available DMEM medium, 125 ml of commercially available F12 medium, 5 ml of non-essential amino acids, 10 ng / ml insulin-like growth factor, 5 μg / ml taurine, 1 μg / ml 5-hydroxytryptamine hydrochloride, 20 μg / ml insulin, 20 μg / ml recombinant transferrin, 4 ng / ml basic fibroblast growth factor, 5 nM triiodothyronine, 1.5 μg / ml hydrocortisone, 3 ng / ml keratinocyte growth factor, 20 ng / ml epidermal growth factor, 50 ng / ml adrenocorticotropic hormone, and 40 μg / ml adenosine. Step 2: In vitro autologous hair follicle tissue In vitro autologous hair follicle tissue was immersed in culture medium A containing 2 U / ml neutral protease and incubated overnight. The next day, the hair follicle tissue was minced and digested with TrypLE at 37°C for 30 minutes, followed by vigorously pipetting. After pipetting, the suspension was filtered through a 100 μm sieve, and the filtrate was centrifuged. After washing three times with phosphate buffer, culture medium A was added to produce a single hair follicle cell suspension. The cell morphology of mixed hair follicle cells in culture medium A is shown in Figure 1. Step 3: Expansion of mixed hair follicle cells The obtained single cell suspension of hair follicles was expanded in two stages using culture medium A and culture medium B. Mixed hair follicle cells were cultured at 5 × 10 4 The cells were seeded at a cell density of 5 × 10 / ml onto a culture dish coated with a substrate for 4 hours, and culture medium A was added. The cells were expanded under conditions of 5% CO2 and 37°C. The liquid was changed every other day, and the cells were passaged when the confluence rate reached 80% or more. After passage, the cells were cultured at a density of 5 × 10 4 The cells were seeded at a cell concentration of / ml onto a culture dish coated with a substrate for 4 hours, and culture medium B was added and expanded under conditions of 5% CO2 and 37°C. The liquid was changed every other day, and when the cell confluence rate reached 80%, the simultaneous expansion culture of hair follicle stem cells, pre-melanocytes, and keratinocytes was completed. The cell morphology of the hair follicle mixed cells in culture medium B is shown in Figure 2. Step 4: Hair follicle mixed cell differentiation and tissue formation 1 × 10 mixed hair follicle cells after expansion culture 4 pieces / cm 2 The cells were seeded at a density onto a substrate-coated culture dish and cultured in culture medium B under conditions of 5% CO2 and 37°C. When the confluence reached 80%, the cells were switched to culture medium C, with the liquid being changed every other day. Hair follicle mixed cells, rich in hair follicle stem cells, pre-melanocytes, and keratinocytes, were induced to differentiate into mature epidermal cells in culture medium C, and the mature cells were arranged in layers. The epidermal cells were tightly bound together by the action of culture medium C, resulting in a tissue structure similar to physiological epidermis, completing the production process. The cell morphology of hair follicle mixed cells in culture medium C is shown in Figure 3. The manufacturing method of Example 1 enabled the economical production of multilayered tissue-engineered skin composed of hair follicle cells, and under these manufacturing conditions, the greatest amount of hair follicle-derived tissue-engineered epidermis was obtained. The morphology of melanocytes in the mixed hair follicle cells is shown in Figure 4. The multilayered tissue-engineered skin composed of hair follicle cells in Example 1 is shown in Figure 5.

[0028] Example 2 Step 1: Prepare the culture medium Preparation of coated substrate: When the volume is 500 ml, the components are 500 ml of commercially available phosphate buffer solution and 50 μg / ml of type IV collagen. Preparation of culture medium A: When the volume is 500 ml, the ingredients are 500 ml of a commercially available keratinocyte medium with clearly defined components, insulin-like growth factor 10 ng / ml, keratinocyte growth factor 3 ng / ml, endothelin 200 ng / ml, melanocyte-stimulating hormone α 50 ng / ml, and gentamicin 50 μg / ml. Preparation of culture medium B: When the volume is 500 ml, the ingredients are 500 ml of a commercially available keratinocyte medium with clearly defined components, 10 ng / ml of insulin-like growth factor, 4 ng / ml of basic fibroblast growth factor, 20 ng / ml of epidermal growth factor, 50 ng / ml of adrenocorticotropic hormone, and 50 ng / ml of melanocyte-stimulating hormone α. Preparation of culture medium C: When the volume is adjusted to 500 ml, the ingredients are 370 ml of commercially available DMEM medium, 125 ml of commercially available F12 medium, 5 ml of non-essential amino acids, 10 ng / ml insulin-like growth factor, 5 μg / ml taurine, 1 μg / ml 5-hydroxytryptamine hydrochloride, 20 μg / ml insulin, 20 μg / ml recombinant transferrin, 4 ng / ml basic fibroblast growth factor, 5 nM triiodothyronine, 1.5 μg / ml hydrocortisone, 3 ng / ml keratinocyte growth factor, 20 ng / ml epidermal growth factor, 50 ng / ml adrenocorticotropic hormone, and 40 μg / ml adenosine. Step 2: In vitro autologous hair follicle tissue In vitro autologous hair follicle tissue was immersed in culture medium A containing 2 U / ml neutral protease and incubated overnight. The next day, the hair follicle tissue was minced and digested with TrypLE at 37°C for 50 minutes (in two separate sessions), followed by vigorously pipetting. After pipetting, the suspension was filtered through a 100 μm sieve, and the filtrate was centrifuged. After washing three times with phosphate buffer, culture medium A was added to produce a single hair follicle cell suspension. Step 3: Expansion of mixed hair follicle cells The obtained single cell suspension of hair follicles was expanded in two stages using culture medium A and culture medium B. Mixed hair follicle cells were cultured at 5 × 10 5 The cells were seeded at a cell density of 5 × 10 / ml onto a culture dish coated with a substrate for 4 hours, and culture medium A was added. The cells were expanded under conditions of 5% CO2 and 37°C. The liquid was changed every other day, and the cells were passaged when the confluence rate reached 80% or more. After passage, the cells were cultured at a density of 5 × 10 5 The cells were seeded at a cell concentration of / ml onto a culture dish coated with a substrate for 4 hours, and culture medium B was added. The cells were expanded under conditions of 5% CO2 and 37°C. The liquid was changed every other day, and when the cell confluence rate reached 80%, the simultaneous expansion culture of hair follicle stem cells, pre-melanocytes, and keratinocytes was completed. Step 4: Hair follicle mixed cell differentiation and tissue formation 8 × 10 mixed hair follicle cells after expansion culture 4 pieces / cm 2 The cells were seeded at a density onto a substrate-coated culture dish and cultured in culture medium B at 5% CO2 and 37°C. When the confluence rate reached 80%, the culture medium was changed to culture medium C, and the liquid was changed every other day. Mixed hair follicle cells, rich in hair follicle stem cells, pre-melanocytes, and keratinocytes, were induced to differentiate into mature epidermal cells in culture medium C, and the mature cells were arranged in layers. The epidermal cells were tightly bound together by the action of culture medium C, resulting in a tissue structure similar to physiological epidermis, completing the production process. According to the manufacturing method of Example 2, multi-layered tissue-engineered skin composed of hair follicle cells can be efficiently manufactured, and under such manufacturing conditions, hair follicle-derived tissue-engineered epidermis can be obtained in only 28 days, which is less than the number of days in Example 1. The quality of the hair follicle-derived tissue-engineered epidermis is the same in the two examples.

[0029] In the above two examples, the ex vivo autologous hair follicles refer to hair follicles isolated from the human body.

[0030] Methods for obtaining extracorporeal autologous hair follicles may be conventional techniques, such as extracting hair follicles using a hair follicle remover. A specific extraction method may be the FUE (follicle unit extraction) method used in hair transplantation. For details, see "Follicular Unit Extraction: Minimally Invasive Surgery for Hair Transplantation." Dermatol Surg. 2002; 28: 720-7. The tip of the hair follicle extractor is pierced into the scalp skin under an electric press, and the hair follicles are extracted. They are then gently extracted with sterile elbow tweezers, with the structure of the hair follicles being completely preserved throughout the entire process.

[0031] In the two examples above, the fabricated multilayered tissue-engineered skin contains melanoblasts and stem cell-like keratinocytes resembling physiological conditions. The multilayered tissue-engineered skin has a stratified structure with a physiological basal layer, spinous layer, and granular layer, with abundant melanoblasts in the basal layer. The tissue-engineered skin has tight intercellular junctions, good toughness, and an abundance of actively dividing basal layer-like cells. The fabricated multilayered tissue-engineered skin can be used to manufacture biomedical materials. It is useful for treating pigmentation defects and epidermal deficiency disorders. For example, when used to treat stable vitiligo, the fabricated multilayered tissue-engineered skin can promote repigmentation of vitiligo and, due to its autologous origin, achieve healing of vitiligo without immune rejection.

[0032] The above description of the embodiments has been provided to facilitate understanding and use of the invention by those skilled in the art. It will be apparent to those skilled in the art that various modifications can be made to these embodiments and that the general principles described herein can be easily applied to other embodiments without creative effort. Therefore, the present invention is not limited to the above embodiments. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention that do not depart from the scope of the present invention are intended to be included within the patentable scope of the present invention.

Claims

1. 1. A method for producing multi-layered tissue engineered skin composed of hair follicle cells, comprising: The method includes the steps of digesting autologous hair follicles in vitro, expanding and culturing the mixed hair follicle cells, and maturing and differentiating the mixed hair follicle cells and forming a tissue structure, In the step of digesting the autologous hair follicles in vitro, The hair follicle tissue is digested by a two-stage enzymatic digestion method, and the hair follicle bulge region, which mainly contains hair follicle stem cells and melanocytes, the outer root sheath, which is rich in keratinocytes, and the hair follicle region, which is rich in dermal papilla cells, are effectively digested to obtain mixed hair follicle cells; In the step of expanding and culturing the hair follicle mixed cells, The hair follicle single cell suspension containing the mixed hair follicle cells was expanded in two stages using culture medium A and culture medium B to obtain the mixed hair follicle cells after expansion culture. In the step of maturation and differentiation of the hair follicle mixed cells and formation of a tissue structure, The expanded hair follicle mixed cells are cultured using culture medium B until they reach a confluence rate of 80%, and then cultured in culture medium C. The culture medium is changed daily during culture in culture medium C, and the hair follicle mixed cells, which are rich in hair follicle stem cells, pre-melanocytes, and keratinocytes, are induced to differentiate into mature epidermal cells in culture medium C. The matured cells are arranged in layers, and the epidermal cells are tightly bound together by the action of culture medium C, forming multilayered tissue-engineered skin composed of hair follicle cells. The culture solution A is When the volume is adjusted to 500 ml, the ingredients are 500 ml of a keratinocyte medium with defined components, 1 to 10 ng / ml of insulin-like growth factor, 0.5 to 5 ng / ml of keratinocyte growth factor, 100 to 500 ng / ml of endothelin, 20 to 80 ng / ml of melanocyte-stimulating hormone α, and 20 to 100 μg / ml of gentamicin; The culture medium B is When the volume is adjusted to 500 ml, the ingredients are 500 ml of a keratinocyte medium with defined components, 1 to 10 ng / ml of insulin-like growth factor, 1 to 10 ng / ml of basic fibroblast growth factor, 10 to 50 ng / ml of epidermal growth factor, 10 to 50 ng / ml of adrenocorticotropic hormone, and 20 to 80 ng / ml of melanocyte-stimulating hormone α, The culture solution C is When the volume is adjusted to 500 ml, the ingredients are 370 ml of DMEM medium, 125 ml of F12 medium, 5 ml of non-essential amino acids, 1 to 10 ng / ml of insulin-like growth factor, 1 to 10 μg / ml of taurine, 1 to 5 μg / ml of 5-hydroxytryptamine hydrochloride, 20 to 80 μg / ml of insulin, 10 to 50 μg / ml of recombinant transferrin, 1 to 10 ng / ml of basic fibroblast growth factor, 1 to 10 nM of triiodothyronine, 0.5 to 5 μg / ml of hydrocortisone, 0.5 to 5 ng / ml of keratinocyte growth factor, 10 to 50 ng / ml of epidermal growth factor, 10 to 50 ng / ml of adrenocorticotropic hormone, and 10 to 50 μg / ml of adenosine, The method for digesting hair follicle tissue using a two-step enzymatic digestion method is as follows: A first enzymatic digestion step and a second enzymatic digestion step, In the first enzymatic digestion step, the in vitro autologous hair follicle tissue is immersed in and incubated in culture solution A containing 0.6 to 2.4 U / ml of neutral protease; In the second enzymatic digestion step, the hair follicle tissue is chopped, TrypLE is added, digestion is performed for 10 to 40 minutes, and then the mixture is vigorously pipetted. After pipetting, the suspension containing the cells and tissue is filtered through a sieve, and the filtrate is centrifuged. After that, a phosphate buffer solution is added to the cell mass, and the mixture is washed 3 to 5 times. After that, culture medium A is added, and a hair follicle single cell suspension containing mixed hair follicle cells is produced.

2. In the step of expanding the hair follicle single cell suspension containing mixed hair follicle cells in two stages using culture medium A and culture medium B, The mixed hair follicle cells were seeded at a cell concentration of 104 to 106 / ml onto a substrate-coated culture dish, and culture medium A was added. The mixture was incubated in 5% CO ２ The cells were cultured at 37°C for 1 hour, and the liquid was changed every other day. When the confluence rate reached 80% or more, the cells were passaged. The cells after passage were inoculated into a substrate-coated culture dish at a cell concentration of 104 to 106 / ml, and culture medium B was added. The mixture was incubated in 5% CO ２ The cells were cultured at 37°C for 1 hour, and the liquid was changed every other day. The method for producing multilayered tissue-engineered skin composed of hair follicle cells according to claim 1, characterized in that upon completion of simultaneous expansion culture of hair follicle stem cells, pre-melanocytes, and keratinocytes, mixed hair follicle cells are obtained after expansion culture.

3. 3. The method for producing multilayered tissue-engineered skin composed of hair follicle cells according to claim 2, characterized in that the substrate for covering the culture dish comprises, as components, 500 ml of phosphate buffer and 10 to 100 μg / ml of type IV collagen when the volume is 500 ml.

4. In the maturation and differentiation of hair follicle mixed cells and the formation of tissue structures, the hair follicle mixed cells after expansion culture were 104 to 105 cells / cm ２ The culture medium was inoculated onto a substrate-coated culture dish at a density of 1000 μg / cm2 and incubated in 5% CO with culture medium B. ２ 2. A method for producing multilayered tissue-engineered skin consisting of hair follicle cells according to claim 1, characterized in that the cells are cultured at 37°C, and when the confluence rate reaches 80%, the culture medium is changed to culture medium C, and the liquid is changed every other day.

Images