KR100648405B1 - Method for isolating primary epithelial cells and reconstructing skin equivalents or dermis equivalents with primary culture cells - Google Patents

Abstract

The present invention relates to a method for separating epithelial cells, characterized in that the human skin tissue or internal organ tissues trypsin / EDTA and at the same time, the self-stirring, according to the present invention, the cell acquisition rate of the isolated epithelial cell population In addition, colony formation efficiency and colony size are significantly increased, and the ratio of hepatocytes is increased, which may be useful for treating damaged human tissues through autograft or allograft.

Description

Method for isolating primary epithelial cells and reconstructing skin equivalents or dermis equivalents with primary culture cells}

1 is a photograph of hematoxylin & eosin staining according to the cell separation process of adult foreskin tissue

2 is a schematic diagram of various epithelial cell separation methods,

3 is a graph showing the cell acquisition rate according to various cell separation methods,

4 is a diagram showing colony formation efficiency according to various cell separation methods,

5 is a graph comparing the relative values of colony forming efficiency and number of colony forming cells according to various cell separation methods,

6 is a graph showing β1 integrin expression by flow cytometry of keratinocytes according to various cell separation methods,

7 is an immunofluorescent staining picture of involukrin of initiating keratinocytes according to various cell separation methods.

Figure 8 is an immunofluorescence staining for involukrin, pan-cytokeratin, α2 integrin of initiating keratinocytes according to various cell separation methods,

Figure 9 is a diagram showing the process of transplanting the keratinocytes isolated in accordance with the present invention in the nude mouse with fibroblasts,

FIG. 10 shows (a) hematoxylin & eosin staining and (b) immunostaining for pan-cytokeratin (c) bimentin of skin structures formed by keratinocytes and fibroblasts implanted in nude mice. Immunostaining for

FIG. 11 shows (a) hematoxylin & eosin staining and (b) immunostaining for pan-cytokeratin, showing that keratinocytes isolated by the cell separation method of the present invention are differentiated into multilayered epithelium on the deepidermal epidermis. ego,

12 is a scanning electron micrograph of fibroblasts after inoculation into BAS ^TM inoculated and cultured for 14 days,

FIG. 13 shows (a) scanning electron micrographs and (b) hematoxylin & eosin staining of fibroblasts after inoculation into isolated epidermal dermal (DED) cells and cultured for 21 days.

14 is a photograph of hematoxylin & eosin staining after inoculation of isolated cultured fibroblasts into artificial dermal structures (Integra ^R , Terumdermis) and cultured for 14 days,

FIG. 15 is a diagram illustrating a process of implanting isolated cultured fibroblasts into artificial dermal structures (Integra ^R , Terumdermis) and incubating for 14 days, followed by implantation into nude mice.

16 is a photograph showing the degree of elevation of the implantation site and 28 days after implanting artificial dermal structures or bioartificial dermal structures and hematoxylin & eosin staining pictures of artificial dermal structures (Integra ^R , Terumdermis),

17 is a photograph and a diagram showing a change in height of the artificial dermis structure or bioartificial dermis structure of FIG.

FIG. 18 is a result of comparing cell growth and division capacity of fibroblasts inoculated on a BAS ^™ by a static method or a dynamic method using cell density.

The present invention relates to a method for separating epithelial cells, and more particularly, to a method for separating epithelial cells, characterized in that magnetic stirring is performed simultaneously with trypsin / EDTA treatment of skin tissues or internal organ tissues.

The skin tissue of the human body is divided into three parts, consisting of the outermost epidermal layer of the skin, the dermis layer below it, and the subcutaneous tissue. The epidermal layer is composed of epithelial cells differentiated into several layers from the basement membrane that allows the epidermal layer and the dermal layer to bind tightly, as well as melanocytes and immune cells.The dermal layer below the epidermal layer is mainly fibroblasts and It consists of several extracellular matrix secreted by cells.

Dermal epithelial cells vary in age and differentiation depending on the layer they are located on. This is because stem cells in the basal layer move to the upper layer while losing the activity of the integrin receptor as cell division is repeated. Epithelial cells go up and further differentiate to reach the top layer, losing the nucleus, leaving only keratin to form a stratum corneum. They are called keratinocytes because the primary function of skin epithelial cells is to protect keratinocytes by making keratin. The stratum corneum periodically drops out of the epidermal layer, and the basal cells divide to compensate for the number of dropped cells, thereby maintaining a constant cell number in the epidermal layer. Cells present in the basal layer are composed of hepatocytes and transit amplifying cells, which are known to be difficult to identify among them. Recently, however, there have been several reports that hepatocyte overexpression of β1-integrin and its high adhesion to the basement membrane are distinguished from daughter cells. β1-integrin overexpressing cells (hepatocytes) are reported to be located above the rete ridge regions of the basal layer and are known to account for about 4-10% of all basal cells (Bickenbach and Chism 1998 ECR). 244: 184-195; Jones and Watt et al 1993 Cell 73: 713-724). When these cells are cultured, they show typical characteristics of hepatocytes such as high colony formation efficiency and slow cell division rate (Bickenbach and Chism 1998 ECR 244: 184-195; Jones and Watt et al 1993 Cell 73: 713-724). ).

Hepatocytes are present in all epithelial tissues in addition to the skin epidermal layer. In the case of the cornea, it is known to exist in the basal layer called the limbus. Hepatic cells are known to be present in the basal layer even in the internal organ tissues of the human body. In the case of mucosal epithelial tissues that form gland structures like the stomach, small intestine, and large intestine, epithelial cells are composed of one layer rather than multiple layers, but even in this case, hepatocytes are known to be located at the deepest part of the gland structure. . That is, hepatocytes of all epithelial tissues can be seen as hiding deeply in areas that are not easily exposed, and the parts where these hepatocytes are located are called stem cells niche. Therefore, hepatocytes are not expected to be easier to separate than other cells.

Skin tissue or internal organ tissues may be damaged by burns, trauma, ulcers, etc., in which case, from the skin or internal organ tissues of the person or others, for the purpose of healing or plasticizing the damaged tissues The method of separating and culturing epithelial (keratin) cells and transplanting them into damaged skin or organ sites is used. To this end, there is an urgent need for a technique for effectively separating epithelial cells. However, only a high proportion of hepatocytes in the isolated cells can ensure a high expansion potential in vitro and the success rate of transplantation.

However, conventionally, the cell separation method developed by Rheinwald and Green (hereinafter, Green's method) has become a common method for the isolation and culture of initial human keratinocytes (Rheinwald and Green 1975). According to this method, epidermal cells are treated with trypsin / EDTA, or trypsin with or without gentle shaking to separate the cells. However, the Green Method provides sufficient cell capture rates for cell separation for research purposes, but insufficient cell separation for histological purposes.

Recently, a two-step enzyme treatment method (hereinafter referred to as thermolysine treatment or disparate treatment), which first separates epidermis-dermis by enzymatic treatment, and then separates epidermal (kerato) cells by enzymatic treatment of epidermis, is introduced (Germain et. al 1993 Burns 19: 99-104, Simon and Green 1985 Cell 40: 677-683). This method involves pretreatment of the skin tissue with thermolysin (Germain et al 1993 Burns 19: 99-104) or dispase (Simon and Green 1985 Cell 40: 677-683) to the epidermis and dermis layers. (dermis) is isolated and then the epidermal layer is separated into individual epidermal cells by trypsin / EDTA treatment. Thermolysine is known to specifically isolate the dermal-epidermal junction of the skin between bullous pemphigoid antigen and laminin sites without destroying desmosomes. Separating the epidermal layer from the dermis by thermolysine or dispensing has the advantage of reducing the chance of fibroblast contamination. However, the two-step enzyme treatment method has a disadvantage in that the action of thermolysine or discontinue cannot be stopped. These enzymes are known to persist for a long time as enzyme-substrate complexes even after removal, which can cause damage to cells even after cell separation is terminated. This possibility has also been demonstrated by the low colony forming efficiency (CFE) of epithelial cells isolated by the two-step enzyme treatment method (see FIG. 4).

Epithelial cells isolated by conventional methods, such as the green method or the two-step enzyme treatment method, have only a limited number of cell propagation during initial culture, and only a part of the transplanted cells after autologous transplantation It is reported to remain, and this is a big problem of epithelial cell transplantation treatment. This is probably due to the low percentage of stem cells contained in the isolated cells. In the case of conventional cell separation methods, it may be because it is difficult to separate basal cells, especially hepatocytes, from the basal membrane only by treatment with enzymes (trypsin) or chelating agents, given the complex reticulated structure and strong binding ability of the basal cells to the basal membrane. do. Tissue specimens of remaining tissue after cell separation demonstrate this, i.e., cells were isolated by these methods and tissue specimens of remaining tissue showed that a significant proportion of basal cells remained inseparable (Figure 1).

Thus, the present inventors have assumed that basal cells will be separated more efficiently if simultaneously applying strong physical agitation in addition to trypsin / EDTA treatment. In addition, it is expected that the acquisition rate of hepatocytes will be significantly increased by this method. That is, the cell separation method was improved by separating the epithelial cells (hereinafter, magnetic stirring method) by applying magnetic stirring using magnetic force in addition to the trypsin / EDTA treatment in the cell separation process. The present inventors also found that the magnetic stirrer method is an efficient method for separating epithelial cells, including an enriched population of stem cells, the magnetic stirrer method and the existing cell separation method, the 'green method', After the separation of epithelial cells from the skin tissue by four cell separation methods, such as thermolysin treatment and dispase treatment, the cell acquisition rate, colony formation efficiency and colony size (cell count / colonies) were determined. Compared. As a result, it was confirmed that the cell acquisition rate, colony formation efficiency and colony size of the magnetic stirring method according to the present invention were significantly increased compared to the other three cell separation methods.

An object of the present invention is to overcome the problems of the conventional cell separation method, to provide a new method for separating epithelial cells with a significant increase in cell acquisition rate, colony formation efficiency and colony size (ratio of hepatocytes). .

In addition, another object of the present invention is to provide a method for producing an artificial artificial skin or an artificial artificial dermal having excellent transplantability using epithelial cells separated by the above method.

Another object of the present invention is to effectively treat damaged skin tissue or internal organ tissue by transplanting the isolated epithelial cells or live artificial skin or live artificial dermis into damaged skin or internal organs due to burns, trauma, ulcers, etc. To provide.

In order to achieve the above object, the present invention provides a method for separating epithelial cells, characterized in that the magnetic or agitation of the skin tissue or internal organ tissues while trypsin / EDTA treatment.

The method of the present invention is a modification of the conventional 'Green's method, to obtain a single cell suspension by applying not only the enzyme action by trypsin / EDTA treatment but also the physical force by strong physical agitation.

In the present invention, the skin tissue or internal organ tissue may be derived from any skin or organ of the animal, but the skin tissue is preferably derived from foreskin or armpit or hip or breast or scalp or pubic or scrotum. Internal organ tissue is preferably derived from the oral mucosa or esophagus or gastrointestinal mucosa or intestinal mucosa or nasal or throat or bronchial or kidney or urethra or uterine mucosa or bladder or kidney or cornea or vagina.

In the present invention, the trypsin / EDTA treatment can be carried out according to well-known green methods (Rheinwald and Green 1975). The amount of trypsin / EDTA to be treated is preferably 0.025-0.25% trypsin and 0.005-0.02% EDTA. If the amount of trypsin / EDTA is lower than this, separation of the cells does not occur well, and if it is high, the number of colonies that survive and damage the cells is markedly reduced.

In the present invention, the magnetic stirring is possible 60 ~ 700rpm for 10 minutes ~ 4 hours, preferably at a speed of 150-500 rpm. In the case of 60 rpm or less, the separation of cells does not occur well, and in the case of more than 700 rpm, the number of colonies viable due to damage to the cells is significantly reduced. The magnetic stirring of the present invention weakens the binding ability of the basal cells to the basement membrane and promotes cell separation.

In order to achieve another object, the present invention is to inoculate the epidermal cells separated by the magnetic stirring method into artificial dermal structures or de-epidermal dermis (DED) alone or simultaneously or sequentially with fibroblasts to provide artificial artificial skin. It provides a method of manufacturing.

In the present invention, the dermal structure may be any commercially available artificial dermal structure, for example, neutralized chitosan sponge or neutralized chitosan / collagen mixed sponge (BAS ^TM manufactured by MTT) or FDA approved or ongoing TransCyte ^™ and Dermagraft ^™ , (Advanced TissueScience) or Integra ^R (Integra LifeSciences) or Apligraf ^R (Organogenesis Inc.) or Graftskin or Alloderm (LifeCell) or Terudermis (Terumo Co.) or Beschitin W (Unitika Ltd.). In addition, the deepidermal dermis (DED) of the present invention is preferably derived from human carcasses or animals.

In the present invention, there is preferably provided a method for preparing a live artificial skin inoculated with melanocytes, hair follicle cells or dermal sheath (dermal sheath) in the artificial dermal structure.

In order to achieve another object, the present invention is to inoculate fibroblasts into artificial dermal structures or deepidermal dermis (DED) to produce a live artificial dermis and transplanted them for the healing of wounds, or to expand or shape tissue Provides a method to implant for the purpose.

In the present invention, the dermal structure can be used in any commercially available artificial dermal structure, the deepidermal dermis (DED) is preferably derived from human carcasses or animals.

In the present invention, there is preferably provided a method for producing live artificial dermis made by inoculating vascular endothelial cells together in artificial dermal structures.

In the method for producing the bioartificial skin or bioartificial dermis of the present invention, the isolated, cultured epithelial cells and / or fibroblasts are inoculated into the dermal construct at a concentration of 1 × 10 ⁴ to 1 × 10 ⁶ cells / cm 2 scaffold. )do. Inoculation method is a dynamic culture (flow) using a shaker (shaking) to attach the cells to the dermal structure (dynamic seeding), and then using a shaker to give a dynamic culture (flow) and the culture method (flow) is known. Both static seeding and static culture were used to attach and culture the cells to the dermal construct.

In order to achieve another object, the present invention is to treat the damaged skin or internal organs by transplanting the epithelial cells isolated by the cell separation method of the present invention alone or in conjunction with the dermal fibroblasts to the damaged skin tissue or internal organ tissue of the animal Provide a way to.

Preferably, the present invention is implanted into the damaged skin tissue or internal organ tissue of the animal, the artificial artificial skin or biosynthetic skin produced by inoculating epithelial cells and / or fibroblasts isolated by the cell separation method of the present invention into artificial dermal structures It provides a method for treating damaged skin or internal organs.

In the present invention, the transplantation method of the isolated cells can be carried out by autologous transplantation or allograft according to methods well known in the art (Wang et al 2000 JID 114: 674-680) (see Example 4).

In the present invention, the skin tissue includes not only the site of skin damage due to burns, trauma, ulcers, etc., but also a site for skin shaping or enlargement of external tissue.

In the present invention, the internal organ tissues include oral mucosa or esophagus or gastrointestinal mucosa or gut membrane or nasal cavity or throat or bronchus or kidney or urethra or uterine mucosa or bladder or kidney or cornea or vaginal region.

Furthermore, the bioartificial skin or bioartificial dermis prepared according to the present invention can be used in various clinical applications or research or test models. For example, models for testing the toxicity or efficacy of cosmetic ingredients; Skin penetration testing or models for testing efficacy or toxicity of a drug; A model for testing the efficacy of a hair growth agent; Models for studying wound healing; Models for studying cell migration or cancer cell infiltration or cancer cell metastasis or cancer progression; A model for studying angiogenesis or a model for testing the efficacy of angiogenesis or angiogenesis inhibitors; It can be used as a model for studying the differentiation of cells or the interaction of epithelial cells with stromal cells and vascular endothelial cells or the function of proteins or genes.

The present inventors have found that the magnetic agitation method according to the present invention, the conventional green method, and the thermolysine treatment method (Germain et al 1993 Burns 19:99) in terms of cell acquisition rate, colony formation efficiency and colony size (cell count / colon) of isolated epithelial cells. -104) and disparate treatment (Simon and Green 1985 Cell 40: 677-683). As a result, the relative values of the cell acquisition rate of the magnetic stirring method with respect to other cell separation methods were 6.3 (green method), 2.2 (thermolysine method) and 4.9 (disparate method) (Figs. 2 and 3); The relative values of colony formation efficiency of the magnetic stirring method with respect to other cell separation methods were 1.2 (green method), 4.2 (thermolysine) and 1.4 (dispase method) (FIG. 4). Since the total number of colony forming cells (hepatocytes) that can be obtained per sample of foreskin is obtained by multiplying the cell acquisition rate by the colony forming efficiency (CFE), the relative value of the magnetic agitation method for the different cell separation methods is 7.2 (Greens), 9.2 (thermolysin) and 6.9 (dispase) (FIG. 5).

In addition, in the magnetic stirring method according to the present invention, the expression level of surface β1 integrin was shifted to the right by the magnetic stirring method (up) (Fig. 6), which is an integrin-bright cell (integrin overexpressing a marker of hepatocytes). Cell population). However, the ratio of involucrin-positive cells, which are markers of terminally differentiating cells, was lower than that of other separation methods (Fig. 7). As a result, the cell separation method according to the present self-stirring method provides improved results in cell acquisition rate and colony formation efficiency, while inhibiting terminal differentiation, thereby reducing the differentiation-aging of cells in vitro and expanding the number thereof. It is the most suitable method for cell separation, and it is also a good method for use in skin transplantation due to the increased ratio of hepatocytes.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to facilitate understanding of the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Cell Isolation and Culture

Initializing keratinocytes were isolated from foreskins of adults. Circumcised foreskin at 4 ° C. in E-medium containing 1% penicillin / streptomycin and 250 ng / ml fungizone (fungizone: Gibco, Cat. No. 15240-062) until the separation procedure is performed. It was left. Cells were isolated within 24 hours after surgery.

The foreskin sample was washed at least eight times in phosphate buffered solution (PBS) containing 5% penicillin / streptomycin. Most subcutaneous tissue was removed from the dermis with sterile surgical scissors and the residual skin was chopped into fine pieces of 1-2 mm square or smaller.

Four methods for separating the cells are (i) the magnetic stirring method according to the present invention, the conventional method (ii) green method, (iii) thermolysin treatment method and (iv) dispase As a treatment method, comparison was performed according to the reference (see FIG. 2).

(i) Magnetic stirring method

Small pieces of tissue were self-stirred at 100 rpm for 30 minutes in 10 ml of 0.125% trypsin and 0.01% EDTA. The isolated cells were washed in 10 ml E-medium containing 20% fetal calf serum to stop the trypsin effect and centrifuge. Cell pellets were resuspended in KGM (Cat No. CC-3111, Clonetics BioWhittaker, Walkersville) and inoculated at 5 * 10 ³ / cm ² . This process was repeated two more times.

(ii) Green's method

Small pieces of tissue were incubated at 37 ° C. for 30 minutes with vortex every 5 minutes in 10 ml of 0.25% trypsin. The isolated cells were washed in 10 ml E-medium containing 20% fetal calf serum to stop the trypsin effect, centrifuged, and the cell pellet was resuspended in KGM (Cat No. CC-3111, Clonetics BioWhittaker, Walkersville). And cultures were inoculated at 5 * 10 ³ / cm ² . This process was repeated two more times.

(iii) Thermolysin Treatment

Small pieces of tissue were treated with thermolysin (250 μg / ml, Cat No. P1512, Sigma-Aldrich Korea) at 37 ° C. for 4 hours. The epidermal layer was separated, washed and further incubated with 10 ml of 0.05% trypsin and EDTA, sometimes with shaking at 37 ° C. for 30 minutes. The isolated cells were washed in 10 ml E-medium containing 20% fetal bovine serum to stop the trypsin effect and centrifuge. Cell pellets were resuspended in KGM (Cat No. CC-3111, Clonetics BioWhittaker, Walkersville) and inoculated at 5 * 10 ³ / cm ² in culture plates.

(iv) Dispase Treatment

Small pieces of tissue were treated with dispase II solution (2.4 U / ml, Cat No. 165859, Roche, Mannheim) at 37 ° C. for 4 hours. The epidermal layer was separated and washed and then further incubated with shaking at times at 37 ° C. for 30 minutes in 10 ml of 0.05% trypsin and EDTA. The isolated cells were washed in 10 ml E-medium containing 20% fetal bovine serum to stop the trypsin effect and centrifuge. Cell pellets were resuspended in KGM (Cat No. CC-3111, Clonetics BioWhittaker, Walkersville) and inoculated at 5 * 10 ³ / cm ² in culture plates.

Cells isolated by four different methods confirmed the cell acquisition rate (see Effect 1 of the invention), and then inoculated onto the coverslip at the density described above to confirm purity by the method of Example 3 (invention). Effect 2), integrin expression was confirmed (see Effect 4 of the invention), or involukrin expression was confirmed (see Effect 5 of the invention). In addition, the cells inoculated in the culture plate confirmed the colony formation efficiency (CFE) after incubation for 2 weeks (see Effect 3 of the invention), or flow cytometry for the ratio of β1-integrin bright cells, the hepatocyte markers, as in Example 2. Or in the same manner as in Example 4 to confirm the presence of differentiation into the skin by transplanting it into nude mice (see Effect 6 of the invention), or inoculated into the dermis deskinned by the same method as in Example 5 to the skin. The differentiation was confirmed (see Effect 7 of the invention).

Example 2: Fluorescence Activated Cell Sorting Method (FACS)

In order to identify the ratio of the β1-integrin-expressing cell group (β1-integrin bright cell), which is known as an indicator of hepatocytes, was compared with the expression level of β1-integrin by fluorescence activated cell sorting method. Cells isolated by four methods were incubated with β1-integrin antibody (chemicon, Cat. No. MAB1959), and then incubated with FITC-bound goat-anti-mouse antibody on ice for 45 minutes. Between each step, cells were washed with PBS containing 5% BSA. At the end of the staining procedure, cells were resuspended at 1 * 10 ⁶ cells / ml and sorted using FACStar ^Plus (Beckton Dickinson). At least 10,000 cells per experiment were analyzed by flow cytometirc analysis. In each experiment, the results were corrected using natural fluorescence and isotype controls (see Effect 4 of the invention and FIG. 6).

Example 3: Immunostaining

Keratin epithelial cells obtained in Example 1 were incubated in the coverslip and then fixed in acetone / methanol (1: 1) at 4 ° C. for 10 minutes. To confirm that the isolated cultured cells consisted only of epithelial cells, the immobilized cells were stained with pan-cytokeratin antibodies, which are markers of epithelial cells (see FIG. 8, Effect 2 of the invention). In addition, the cells were stained with α2 integrin antibody (see FIG. 8, effect 4 of the present invention) to confirm that the cultured cells exhibited the characteristics of basal cells, and stained with the involukrin antibody to confirm the percentage of cells differentiated. (See FIG. 8, effect 5 of the invention). Integrin α2 (chemicon), mouse monoclonal antibody against β1 (chemicon), and pan-cytokeratin (1:10, Novocastra, marker of keratinocytes), involucrin; Biomedical Technologies, 1 Rabbit polyclonal antibody against: 50, differentiation marker of keratinocytes). After incubation with the initiating antibody, standard ABC staining was performed using kit (Vector Laboratories).

Example 4: Differentiation of keratinocytes implanted into nude mice into skin

Nude mice were implanted to assess in vivo the differentiation of isolated human keratinocytes into perfect skin (see FIG. 9, Effect 6 of the invention). First, a full-thick wound with a diameter of 1 cm is made on the back of a nude mouse, and then a plastic chamber is implanted in the wound. In a chamber in which a cell suspension in which keratinocytes (5 × 10 ⁵ cells / cm ² ) and dermal fibroblasts (1 × 10 ⁵ cells / cm ² ) cultured in Example 1 were mixed with KGM was previously implanted into the back of a nude mouse. Inoculation. After one week the plastic chamber was removed to induce differentiation of the epidermis. The regenerated skin tissue was separated, fixed in 3.7% formalin / phosphate buffer solution, and subjected to hematoxylin & eosin and necessary tissue staining to confirm the differentiation of the inoculated cells into the skin (see FIG. 10).

Example 5: Differentiation of keratinous epithelial cells into the stratified skin epidermal layer from deepidermal dermis (DED)

In vitro evaluation of the differentiation of cells and fibroblasts into complete skin was inoculated in de-epidermized dermis (DED) obtained by removing the epidermis from human carcasses and incubated for 3 weeks ( 11, effect 7 of the invention). Dermal fibroblasts were inoculated at a density of 1 × 10 ⁵ cells / cm ² to the bottom of the DED (dermal reticulus) and one day later keratinocytes at ^a density of 1 × 10 ⁵ cells / cm ^{2 to the} dermal papillarus. Incubated for 1 week in water and 2 weeks at E-medium at the air-liquid interface. It was fixed in 3.7% formalin / phosphate buffer solution, and hematoxylin & eosin and necessary tissue staining were performed to confirm the differentiation of the inoculated cells into the skin.

Example 6-7

The artificial artificial skin may or may not contain fibroblasts constituting the dermal layer. For reconstruction of artificial skin including fibroblasts, the fibroblasts of the dermal layer are separated and cultured and inoculated directly into the living body to form a dermal layer. 9, 10, effect 6 of the invention, or inoculated into the artificial dermis in vitro to produce a live artificial dermis (Fig. 11, 12, 13, 14, effect 7 of the invention), or transplanted in vivo (FIG. 15, effect 8 of the invention).

Example 6 Inoculation of Fibroblasts Separated and Cultured in Artificial Dermal Structure

Cut the dermis of the foreskin with scissors by the method of Example 1 (self-stirring method, Green's method), or separate the dermis layer (thermolysine method, disperse method) after thermolysine or dispase treatment in 10 ml of 0.07% collagenase solution. Dip and incubate for 2 hours at 37 degrees Celsius and pipette to obtain fibroblasts. The fibroblasts thus isolated were inoculated into artificial dermal structures immediately after incubation in F-media (DMEM: F-12 = 3: 1, 10% FBS, 1% penicillin-streptomycin), or frozen stock solution (DMEM 50%, Fetal calf serum 40%, DMSO 10%) and thawed as needed to inoculate the dermal structures. The dermal structures are perforated in sterile hoods with a diameter of 8mm to 100mm and placed in a 24 well and 100mm diameter dish. In the preparation of 8mm diameter artificial dermis, 1 × 10 ⁵ viable cells (determined as trypan blue solution) were diluted with a minimum volume of DMEM culture solution to dermal structures (MTT's Bioartificial skin (BAS ^TM ) (FIG. 12, effect 8 of the invention). Or TransCyte ^™ and Dermagraft ^{™ from} Advanced TissueScience, or Integra ^R from Integra LifeSciences (see FIG. 14, Effect 8 of the invention), Apligraf ^{R from} Artificial skin or Organogenesis Inc., Alloderm from Graftskin or LifeCell or Terumo Co. Japan. To Terudermis (FIG. 14, Effect 8 of the invention), or Beschitin W of Unitika Ltd., Japan, de-epidermized demis (DED) (FIG. 13, Effect 8 of the invention). Inoculate them evenly. After 3-5 hours at 37 ^° C. with 5% CO ₂ , 50 μl of DMEM is added. After 24 hours, 1 ml of medium is added to each well. The biosynthetic dermis is maintained under the same conditions for 3-4 weeks in order to achieve a constant congestion, and the culture medium is exchanged three times a week to prepare the biosynthetic dermis.

Example 7 Implant Experiments on Nude Mice of Raw Human and Human Dermis

In the same manner as in Example 6, Integra ^R and Terudermis were inoculated with fibroblasts, which were made by injecting fibroblasts or Integra ^R and Terudermis (artificial dermis) into a nude mouse's back and confirming the effect on tissue expansion (FIG. 15). , 16, effect 9 of the invention). Nude mice are bred in cleanrooms. A 1 cm long incision is made horizontally on the back of the nude mouse. Using tweezers, implant the raw artificial dermis or artificial dermis of diameter 8mm directly over the fascia, sew it with a surgical suture, and apply with sterilized gauze. To prevent infection, antibiotics of empicillin and streptomycin are added to the drinking water. The height of the implant site of the test animal is examined daily and killed after 28 days. Tissues containing both normal skin and implant sites are separated from the test animals and used as a biopsy sample. Samples were fixed with 3.7% formalin / phosphate buffer solution, prepared with paraffin blocks, and sectioned and stained with hematoxylin & eosin solution.

1. Cell acquisition rate

The cells were separated in four ways, and the remaining tissues were fixed and hematoxylin & eosin stained to identify the cells that were not separated. While the cells were separated by the magnetic stirring method, there were almost no cells remaining in the remaining tissues, while the cells were separated by the other separation methods and many basal cells remained in the remaining tissues (FIG. 1). Complete separation of such cells is an effect by magnetic stirring, which was further verified by measuring the number of isolated cells (Table 1, Figures 2, 3). The magnetic stirring method according to the present patent shows an increase in cell acquisition rate of about 700% compared to the green method.

Table 1. Cell Acquisition Rate-Total Cells per Poppy (X 10 ⁷ )

Way Magnetic stirring Green way Thermolysine Law Disparate method Average ¹ 4.24 ± 0.57 0.68 ± 0.07 1.97 ± 0.51 0.87 ± 0.30 range 2.34-6.76 0.60-0.88 0.36-3.25 0.11-2.24

1: Mean ± SEM

2. Purity of Cells

As a result of fluorescent staining of the isolated cultured cells using pan-cytokeratin, an antibody of keratinocytes, it was intended to confirm the presence of other types of contaminated cells in the isolated cultured cells. From the fact that the ratio of the pan-cytokeratin-positive cells (epithelial cells) was 100%, it was confirmed that the cells cultured in isolation were pure keratinocyte groups without fibroblasts (FIG. 8). There was no difference in the ratio of pan-cytokeratin-positive cells in cells cultured by different cell separation methods. Thus, the purity of cells cultured by magnetic stirring is shown to be the same as other separation methods.

3. Colony Formation Efficiency (CFE)

The presence of hepatocytes can be confirmed by colony-forming efficiency (CFE). CFE of the keratinary epithelial cell group isolated by the magnetic stirring method was the highest (Table 2, Figure 4). In particular, the formation frequency (CFE) of large colonies (> 128 cells / colonies) showed a much higher increase (Table 2). These results indicate that the proportion of hepatocytes is significantly increased in keratinocytes isolated and cultured by magnetic stirring.

Table 2. Colony Formation Efficiency (%) ¹

Colony Magnetic stirring Green way Thermolysine Law Disparate method <32 0.979 ± 0.419 0.416 ± 0.177 0.265 ± 0.123 0.571 ± 0.136 > 32 1.149 ± 0.319 0.947 ± 0.345 0.275 ± 0.122 0.826 ± 0.298      32-100 0.485 ± 0.122 0.488 ± 0.199 0.163 ± 0.076 0.419 ± 0.169      > 100 0.672 ± 0.213 0.461 ± 0.147 0.112 ± 0.048 0.407 ± 0.140

1: A total of 10,000 cells were seeded in 6-well plates and incubated for 2 weeks.

Therefore, cells isolated by the magnetic stirring method of the present invention showed higher values than other cell separation methods in terms of colony formation efficiency (CFE) and cell acquisition rate. Therefore, it can be seen that the physical force applied through the magnetic stirring increases the cell acquisition rate and colony formation efficiency. As a result, according to the present invention, the total number of colony forming cells per foreskin sample increased more than 9 times (FIG. 5).                     

In addition, the low intake rate of skin grafts in adult patients caused by the lack of hepatocytes in culture may be complemented by the methods of the present invention.

4. Integrin Expression

As a result of immunostaining, all the keratinocytes cultured in the isolated culture express α ₂ integrin expressing only cells in the basal layer (basal cells) (FIG. 8). By epithelial cell division.

Flow cytometry of β1-integrins shows the relative proportion of β1-integrin-bright cells, markers of hepatocytes, in the cutaneous keratinocytes cultured by each method. The distribution of β1-integrin-bright cells in the keratinocytes isolated and cultured by the magnetic stirring method according to the present invention is shifted to the right, indicating that the proportion of hepatocytes is the highest in the cell population isolated by this method. (FIG. 6).

5. Involukrin Expression

Involukrin, a differentiation marker of keratinocytes, is always expressed in cultured epithelial cells at a low frequency (Fig. 8). The ratio is 7% in the magnetic stirring method, 7% in the green method, 17% in the thermolysine method, and the disparate. 23% in the law (Table 3, FIG. 7). Cells expressing involuclin die by differentiation-aging. Therefore, the keratinocyte epithelial cells separated and cultured by the self-stirring method and the green method according to the present invention have a 40% increase in the number of cells due to differentiation and aging due to differentiation and aging. More advantageous to.

Table 3. Proportion of Involuclin Expression

Magnetic stirring Green way Thermolysine Law Disparate method Involukrin + Cells 7 ± 2 (%) 7 ± 1 (%) 17 ± 2 (%) 23 ± 6 (%) P-value <0.005 <0.05

6. In vivo differentiation of keratinocytes

Suspensions of dermal keratinocytes and dermal fibroblasts implanted into the back of nude mice also formed complete skin consisting of the epidermis, dermis and basement membrane (FIG. 10). Epithelial cells were positive for human-specific pan-cytokeratin, and dermal cells were positive for human-specific bimentin, indicating that these epithelial cells and fibroblasts were both human-derived and transplanted cells. Can be. In addition, it can be seen that these epithelial cells and fibroblasts surviving at the wound site of nude mice moved together to differentiate into epidermal and dermal layers, respectively.

7. In vitro differentiation of keratinocytes

In nature, keratinocytes differentiate into stratified multilayered epidermis. In order to examine the differentiation ability of the keratinocytes separated and cultured into the multi-layered epidermis by the self-stirring method of the present invention, three weeks after direct inoculation into the deepidermal dermis (DED), the tissues were fixed and hematoxylin & eosin staining. It was. As a result, positive epithelial cells formed a multilayer for pan-cytokeratin (FIG. 11).

8. Live artificial dermis prepared by inoculating fibroblasts into artificial dermal structures

When using the dynamic inoculation provided at the time of inoculation and incubation of the flow, the number of fibroblasts attached to the BAS was increased as compared with that of the static inoculation (FIG. 16). Scanning electron micrographs of fibroblasts inoculated with BAS confirm that the attached cells are abundantly secreting extracellular epilepsy (FIG. 12). Therefore, it can be seen that the cells of the live artificial dermis are functionally the same as in vivo. On the other hand, fibroblasts inoculated with DED showed a degree of cell congestion similar to that found in the deep dermis and relatively evenly distributed in the BAS compared to the cells mainly attached to the surface only in the BAS (Fig. 13). Fibroblasts inoculated in Integra and Terumdermis also show similar congestion and even distribution of cells as in DED (FIG. 14).

9. Structure of Live Human and Human Dermis Implanted in Nude Mouse

14 days after fibroblasts were inoculated with Teruthemis and Integra (figure Dermis) (FIG. 14), or 28 days after transplanting Teruthemis and Integra directly (nucleus) to nude mice (FIG. 15) The artificial artificial dermis or artificial dermis was examined by hematoxylin & eosin staining (FIG. 15, 16). No signs of inflammatory reactions were observed at the graft site or surrounding tissues, and the graft site was well fused with the surrounding tissues, and much of the original size remained intact (FIG. 16). Influx of fibroblasts and blood vessels was found throughout the graft site, and fibroblast congestion at the graft site was similar to that in the mouse dermis (FIG. 16). The height of the graft was measured using a caliber, but the height of the graft was smaller than the measurable range, and the height was measured using a stained photograph of the tissue specimen (FIG. 16). Both Teruthemis and Integra occurred volume reduction after transplantation, which is thought to be due to contraction of collagen and degradation of the implant. Cell-inoculated live artificial dermis had a smaller volume reduction in implants than artificial dermis and Integra had a Teruthemis (see Table 4).                     

Table 4. Height change after 28 days of implantation of artificial and live artificial dermis

Height of transplantation site ¹ Full height ² The Ruther Miss Integra The Ruther Miss Integra Artificial dermis 0.5-0.6 0.6-0.7 1.4-1.5 1.5-1.9 Raw artificial dermis 0.7-0.9 0.8-0.9 1.5-2.1 1.7-2.1

1: Relative value of dermal structure height 28 days after transplantation to original height of dermal structure (1). 2: Relative value of dermal structure height 28 days after transplantation to height 1 from fascia to stratum corneum of nude mouse.

 On the other hand, the artificial artificial skin or artificial artificial dermis according to the present invention can be used even when the wound is large, such as burns, or when the movement of cells around the wound is difficult, such as diabetes, and can be used immediately for regenerating recessed tissue such as molding. Skin or live dermis can be obtained.

Claims (14)

A method of separating epithelial cells from the skin or internal organs with trypsin or trypsin / EDTA and simultaneously stirring, wherein the trypsin is treated with a trypsin amount of 0.025 to 0.25%, and trypsin / EDTA. When the treatment is treated with an amount of trypsin of 0.025 to 0.25% / EDTA of 0.005 to 0.02%, the magnetic stirring is a method for separating epithelial cells, characterized in that stirring for 10 minutes to 4 hours at a speed of 60 to 700rpm . The method of claim 1, wherein the skin tissue is derived from the foreskin, armpits, hips, breasts, scalp, cornea, pubic part or scrotum. The method of claim 1, wherein the internal organ tissue is derived from oral mucosa, esophagus or gastrointestinal mucosa, mucosa, nasal cavity, throat, bronchus, kidney, urethra or uterine mucosa, bladder, kidney, or vagina Separation Method. delete delete delete Separating epithelial cells from skin tissue or internal organ tissues by the method of any one of claims 1 to 3; And to the artificial artificial dermis prepared by inoculating fibroblasts into artificial dermal structures, de-epidermal dermis (DED), or artificial dermal structures or de-epidermal dermis (DED) alone or together with fibroblasts. Method of producing artificial artificial skin comprising the step of inoculating. A method of manufacturing artificial skin structures comprising inoculating melanocytes into the artificial artificial skin prepared by the method of claim 7. A method of manufacturing an artificial skin structure comprising the step of inoculating hair follicle cells or dermal sheath on the live artificial skin prepared by the method of claim 7. A method of manufacturing an artificial skin structure comprising inoculating vascular endothelial cells into a living artificial skin prepared by the method of claim 7. delete delete delete delete

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