KR100927374B1 - Skin cell co-culture method and cell therapeutic composition using same - Google Patents

Abstract

Provided are a skin cell co-culture method of mixing and culturing keratinocyte, melanocyte, fibroblast, and endothelial cell which are extracted from minimal donor sites and a cell therapeutic composition using the same. Since various types of skin cells extracted from one type of donor site are mixed and co-cultured, due to a paracrine effect between cells, all the cells can be well grown even in a serum-free medium. In addition, the cells can be well grown in a medium that is not a medium specified for culture of each type of cells. In addition, the cells can be well grown even in a case where the number of cells is too small to be well grown in a separate culture. In addition, unlike the separate culture where freezing storage and thawing of melanocyte is almost not available, the mixed co-cultured cells can be well grown after the freezing storage and thawing. In addition, expression of an SMA (smooth muscle actin) which is induced in an in-vitro fibroblast culture is not induced in the mixed co-culture, so that fibroblast can be cultured without induction of differentiation. A cell therapeutic including the mixed, co-cultured skin cells can show an effective wound healing capability in comparison with conventional cell therapeutics including only the keratinocyte.

Description

Co-culture method of skin cells and cell therapy composition using the same}

The present invention relates to a method for coculture of skin cells, and more specifically, to the culture of epidermal cells (Keratinocyte), melanocytes (Melanocyte), fibroblasts (Fibroblast) and endothelial cells isolated from the minimum donor site It relates to a skin cell co-culture method and a cell therapeutic composition using the same.

Until now, cell culture technology has only been in the step of separating and culturing one cell at a donor site of less than 1 cm ² , and suffered from the cultivation itself due to the lack of separated cells. Various wound tissues, including burns, are difficult to recover with cell therapies that consist of only one cell, and the effects are greater when various cell groups on the skin act on wound healing. Therefore, keratinocyte, fibroblast, and melanocyte are simultaneously separated and mixed co-culture at the minimum donor site to promote the culture through exchange between cells without supply of cytokine or growth factor that must be added from the outside during single culture. It is possible to create an environment and maintain an environment similar to the actual skin tissue.

In the case of the mixed co-cultured cells, attachment was faster after tissue separation than in the cultured cells alone, and fibroblasts did not express α-smooth muscle actin, which was observed in the culture alone. This indicates that differentiation is not induced in the case of mixed co-culture during the culture, and it can be seen that an environment similar to the in vivo environment is formed. The method of mixed co-culture of two or more living cells according to the present invention will be useful for developing a multicellular therapeutic agent that can promote wound healing to the maximum.

Accordingly, a main object of the present invention is to provide a co-culture method of mixing two or more skin cells separated at the minimum donor site in one medium.

Another object of the present invention to provide a cell therapy composition comprising the skin cells cultured by the co-culture method.

According to an aspect of the present invention, the present invention is a mixture of isolated epidermal cells (Keratinocyte), melanocytes (Melanocyte), fibroblasts (Fibroblast) and endothelial cells (Endothelial cells) in serum-free media (serum-free media) It provides a skin cell co-culture method characterized in that the culture.

In the present invention, preferably, the cells are co-culture method of skin cells, characterized in that separated from one donor site (donor site). More preferably, the donor site is characterized by using autologous skin tissue of the patient to transplant the cocultured skin cells. According to the present invention, it is possible to effectively co-culture various skin cells isolated from the minimum donor site of 0.5-2 cm ² size.

In the present invention, preferably, the epidermal cells (Keratinocyte) and melanocytes (Melanocyte) are separated from the epidermal layer of the donor site, and the fibroblast and endothelial cells are the dermal layer of the donor site. It provides a skin cell co-culture method characterized in that separated from.

In the present invention, it is preferably mixed cultured by the paracrine effect between cells without supplying the cytokine (cytokine) or growth factor (growth factor) necessary for single culture in the serum-free medium. It provides a skin cell co-culture method. This paracrine effect allows all cells to grow well in a serum-free medium and to grow well in a medium that is not a specialized medium for culturing each cell.

Serum-free medium (KGM: Keratinocyte Growth Medium) used in the examples of the present invention is a medium originally used for culturing epidermal cells. Therefore, the culture of other cells alone is not well cultured. Fibroblast cultures require bFGF to prevent differentiation and melanocytes require bFGF, insulin, PMA, hydrocortisone, etc., but PMA is known to be carcinogenic. The serum-free medium of the present invention is not a medium in which growth factors or cytokines are completely excluded, but co-cultivation is possible even in a situation where factors such as bFGF and PMA, which are necessary for each single culture, are excluded.

 In the present invention, preferably the ratio of epidermal cells + melanocytes: fibroblasts + vascular endothelial cells is 15: 1 to 25: 1, most preferably 20: 1 skin cells, characterized in that the mixed culture Provide a coculture method. In the embodiment of the present invention, a number of co-culture tests at various magnifications showed that all cells grew best at a ratio of 20: 1 (epidermal cells + melanocytes: fibroblasts + vascular endothelial cell). If the number of epidermal cells is more than the above range, the lack of fibroblasts does not produce enough ECM components, so the adhesion of epidermal cells is not good. Since the cells must be grown through mass exchange, it was confirmed that clustering of fibroblasts appeared when cultured in serum-free medium.

In the present invention, it provides a skin cell co-culture method, characterized in that the expression of smooth muscle actin (SMA) which is preferably induced in the culture of fibroblasts alone is not induced. Not expressing the SMA (smooth muscle actin) means that the differentiation-induced fibroblasts can be cultured.

In the present invention, preferably, melanocytes that do not grow well after cryopreservation and thawing during culturing alone provide a method for co-culture of skin cells, characterized in that they grow well even after cryopreservation and thawing. Melanocytes were not stored (frozen storage) in nitrogen tanks when cultured alone and almost all died upon culturing again, but when mixed co-cultured according to the present invention, they grew well even after freezing storage and thawing.

According to another aspect of the present invention, the present invention comprises epidermal cells (Keratinocyte), melanocytes (Melanocyte), fibroblasts (Fibroblast) and endothelial cells (Endothelial cells) co-cultured according to the co-culture method of the present invention It provides a wound healing cell therapy composition. The wound healing means to heal or alleviate the symptoms of hypopigmentation or vitiligo caused by a wound or a loss of melanocytes or a decrease in melanin production due to trauma or burn of the skin. The cell therapy agent of the present invention showed superior wound healing ability than the conventional cell therapy agent that separates and cultures only epidermal cells (Keratinocytes) and sprinkles or attaches to the burn site.

The cell therapy compositions of the present invention may be prepared in the form of formulations common in the art, such as injections, sprays, matrices, or films, which may be surgically implanted directly into the skin injuries, or in the form of sprays. It can be applied to, or applied to, the skin injury in the form of a matrix or film, or transferred to the skin injury after administration to a vein. Cells co-cultured in the composition of the present invention may vary depending on the type of disease, route of administration, age and sex of the patient, and degree of disease, but preferably 10 ⁴ to 10 ⁸ cells for the average adult.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1.Isolation and Culture of Primary Cells from Skin Tissue

About 1 cm ² of foreskin obtained by surgery is cut into small tissues of 5 mm 2 or less and washed 10 times in cold D-PBS containing 5% penicillin-streptomycin (PS). Add Dispase (P-3417, Sigma) and perform enzymatic reaction in 37 ℃ water bath for 2 hours, transfer to 100㎜ dish containing 10ml 5% PS-PBS, and separate epidermal and dermal layers with tweezers.

In order to separate epidermal cells (Keratinocyte) and melanocytes (Melanocyte), transfer the separated epidermal layer into a 15ml tube, add Trypsin-EDTA, and react for 1 hour in a 37 ℃ water bath. Put a 100㎛ cell strainer on a 50ml tube and quench the Trypsin-EDTA reaction solution. To stop Trypsin-EDTA action, add the same amount of FBS and centrifuge for 5 min at 1,200 rpm, 4 ° C. Remove the supernatant and precipitate the epidermal cells (Keratinocyte) and melanocytes (Keratinocyte Growth Medium: KBM (Keratinocyte Basal Medium, 2ml BPE, 500µl Insulin, 500µl hEGF, 500µl GA) -1000, 500µl Hydrocortisone (Cambrix, USA) is added to the suspension and the cell number is leaked.

To separate the fibroblast and endothelial cells, cut the separated dermal layer with finely cut scissors, transfer to 50ml tube, add 0.1% collagenas, and react for 1 hour in 37 ℃ water bath. To remove the collagenase enzyme reaction, FGM (Fibroblast Growth Medium media: FBM (Fibroblast Basal Medium), 10ml FBS, 500µl Insulin, 500µl rhFGF-B, 500µl GA-1000) was added at 1,200rpm, 4 ℃, 5 For two minutes, the supernatant is removed by centrifugation twice.

Example 2 Mixed Culture of Epidermal Cells (Keratinocyte), Melanocytes (Melanocyte), Fibroblasts, Endothelial Cells

Epidermal cells (Keratinocyte), melanocytes (Melanocyte) and the fibroblasts (Fibroblast) and Endothelial cells (Endothelial cells) isolated from the epidermal layer is isolated from the epidermal layer in the initial primary culture in 20: 1 ratio. In the initial separation of cells from the epidermal cell, epidermal cells and melanocytes are separated from the epidermal layer and fibroblasts and vascular endothelial cells are separated from the dermal layer. When the ratio of fibroblasts and vascular endothelial cells is 20: 1, it was confirmed by the test results that the most favorable for co-culture. Subcultures were allowed to react with Trypsin-EDTA for 3 minutes to separate melanocytes, fibroblasts, and vascular endothelial cells, except for epidermal cells, from the culture dish, followed by further reaction for 10 minutes to separate epidermal cells. In each Trypsin-EDTA reaction solution, FBS was added to stop the enzyme reaction, centrifugation to remove the supernatant, and a cell suspension was made to drain the cell number. 3 × 10 ⁵ epidermal cells and a cell suspension containing melanocytes, fibroblasts, and vascular endothelial cells at a ratio of 1/20 ( ^1.5 × 10 ⁴ ) are mixed and cultured in KGM. In the secondary passage culture, melanocytes, fibroblasts and vascular endothelial cells, which are separated within 3 minutes by trypsin-EDTA treatment, are separated together, and epidermal cells separated after 10 minutes of treatment are separated. Therefore, in the case of subculture, the ratio of epidermal cells: fibroblasts + melanocytes + vascular endothelial cells was found to be most advantageous in co-culture. In the same manner, cell culture and cell number were observed while subcultured to passage number 4. 1 is a photograph observing the cell shape in the primary culture (P = 1) and subculture (P = 2 to 5) mixed culture in accordance with the present invention.

TABLE 1 Cell number in primary culture (P = 1) and subculture (P = 2 ~ 5)

Epidermal cells Melanocytes Fibroblast Vascular cells Primary culture 1.80E + 06 8.00E + 05 P2 6.80E + 05 7.40E + 05 P3 4.24E + 06 1.95E + 06 P4 1.04E + 06 3.00E + 05 P5 2.40E + 05 1.55E + 05

In contrast, when the fibroblasts (1x 10 ⁶ cells) and epidermal cells (1x 10 ⁶ cells), which were initially isolated from the foreskin tissue, were cultured in KGM medium alone, cell shapes were observed. 2A and 2B are photographs when the fibroblasts and epidermal cells initially isolated from the foreskin tissue were cultured alone in KGM medium. In A. fibroblasts did not grow healthy in serum-free KGM and showed a tendency to aggregate together. In B., epithelial cells alone did not attach to the culture dish at all. 2C is a photograph of co-culture of epidermal cells + melanocytes (1x 10 ⁶ cells): fibroblasts + vascular endothelial cells (5x 10 ⁴ cells) in KGM at a ratio of 20: 1. When co-cultured according to the present invention, both cells grew well. DEF of Figure 3 is a test result by varying the mixing ratio of the cells. The ratio of epidermal cells + melanocytes: fibroblasts + vascular endothelial cells was compared with 10: 1 (D), 20: 1 (E), and 30: 1 (F), respectively. Cell observation revealed that all cells were best cultured at a 20: 1 ratio.

Mixed co-cultivation is more advantageous in culturing in serum-free media (KGM) because it is possible to culture cells by mutual exchange between cells without supply of cytokine or growth factor from the outside. In the case of media), as it increases only the culture rate of cells promoted by serum, such as fibroblasts, the condition of other cells may worsen, resulting in a breakdown of the division rate of cells. To demonstrate this, 10% serum-added E-media (Sigma, Poole, U.K.) was used alone or mixed culture. As shown in FIG. 4, in the medium to which serum was added, both epithelial cells alone and epithelial cells and fibroblasts were not cultured properly, but both were cultured in serum-free medium (KGM). it was good.

Example 3. Storage and Thawing of Mixed Co-Cultured Cells

Epidermal cells: Fibroblasts, melanocytes, and vascular cells with a ratio of 4: 1 or 20: 1, suspended in freezing media (KGM: FBS: DMSO = 5: 4: 1) at a total cell count of 1 × 10 ⁶ cells. Leave in a freezer for one day, put in liquid nitrogen the next day and stored for a long time. 10 days after storage, the cells were dissolved again, and then inoculated in a culture plate to confirm cell viability and cell status, and fluorescence staining confirmed that each cell normally survived. 5 is a photograph confirming cell viability and cell state after cryopreservation and thawing of mixed co-cultured cells according to the present invention. From this, melanocytes (HMB45), which did not grow well after cryopreservation and thawing, were found to grow well after cryopreservation and thawing.

Example 4. Immunofluorescence Staining of Mixed Co-Cultured Cells

For identification of mixed co-cultured cells, markers expressed only in each cell were measured by immunofluorescence staining. Epidermal cells: Fibroblasts + melanocytes + vascular endothelial cells were fixed by co-cultured cells at a ratio of 20: 1 with cold methanol for 10 minutes at 4 ° C, followed by PBS with 0.2% TritonX-100. Permeabilization by reacting while shaking at room temperature for 10min. After washing 3 times with 5 min shaking in PBS, the reaction was performed at room temperature for 1 hour with PBS added with 20% NGS (normal goat serum), and cytokeratin 14, HMB45, which are markers for epidermal cells, melanocytes, fibroblasts, and vascular endothelial cells. Reaction with vimentin, vWF (Von Willebrand Factor) antibody at room temperature for 2 hours. After washing three times by shaking for 5 minutes in PBS, the secondary antibody (Cy2, Texas Red) to the fluorescent dye is reacted for 1 hour 30 minutes at room temperature. After washing 3 times by shaking for 5 minutes in PBS, and then reacted for 5 minutes in DAPI (Sigma D-9542) for nuclear staining, and mounted with Vectashield (Vector Lab.). Finally, Leica's MacroFluo ^TM fluorescence microscope was used. 6 is a photograph of immunofluorescence staining of cells co-cultured with passage number 2. Red arrow: Melanocyte, Blue arrow: Fibroblast. Immunofluorescence staining of mixed coculture cells of passage number 2 shows that melanocytes (HMB45) are present. 7 is a photograph of immunofluorescence staining of cells co-cultured with passage number 2. Immunofluorescence staining of cells co-cultured with passage number 2 shows that vascular endothelial cells (vWF) are present. Fig. 8 shows immunofluorescence staining of mixed co-cultured cells of passage number 3. Red arrow: Melanocyte, Blue arrow: Fibroblast. Immunofluorescence staining of the mixed coculture cells of passage 3 showed that melanin cells (HMB45) and epidermal cells (CK14) exist. 9 is a photograph of immunofluorescence staining of cells co-cultured with passage number 3. Immunohistofluorescence staining of passage co-cultured cells of passage 3 indicates that melanocytes (HMB45), epidermal cells (CK14) and fibroblasts (vimentin) are present. 10 is a photograph of immunofluorescence staining of cells co-cultured with passage number 4. Red arrow: Melanocyte, Blue arrow: Fibroblast. Immunofluorescence staining of cells co-cultured with passage number 4 shows that melanocytes (HMB45) are present.

11 is a photograph confirming that α-SMA (α-smooth muscle actin) is expressed in mixed co-cultured cells according to the present invention. Fibroblasts alone culture (p = 2) Except for the figure, all of the results were co-cultured at a ratio of 20: 1 (epidermal cells: fibroblasts + melanocytes + vascular endothelial cells). Α-Smooth muscle actin (α-SMA) expressed in the culture of fibroblasts alone was not expressed in the mixed co-culture. Therefore, it can be seen that the mixed co-culture according to the present invention inhibits the differentiation of fibroblasts.

Example 5. In vivo skin formation test of mixed co-cultured cells

In order to prevent the suspension of the mixed co-cultured cells prepared according to Example 2 and to prevent the rapid healing of nude mouse tissue, a 3 ml syringe handle was cut to an appropriate size (1 cm in height) to prepare a syringe splint. do. Anesthetize the nude mouse, cut the skin on the back and insert the syringe split. Secure the syring split and purse-string suture to prevent cell culture fluid leakage. Add the mixed coculture suspension. To prevent contamination from outside and maintain the skin environment, Tegaderm (3M) was added and antibiotics were administered for 7 days. Teraderm and syringe splint were dropped 5 days after the procedure and the wound was left open for a day. Figure 12 is a photograph observing the in vivo skin formation ability in nude mice of mixed co-cultured cells according to the present invention.

Example 6 Immunostaining of Skin Tissues Formed In Vivo

In vivo skin formation test of mixed co-cultured cells is performed, and nude mice are sacrificed, and the formed tissues are fixed in formalin to prepare paraffin tissue sections. After deparaffinization and hydration, pretreatment procedures for each antigen are performed for antigen antibody immunostaining. To confirm epidermal and dermal layer formation, multi-cytokerain and vimentin antibodies were reacted, and cytokeratin 14 and type IV collagen antibodies were reacted to confirm basal layer formation and basement membrane formation. HMB 45 antibody was reacted to confirm the location of melanocytes and melanin formation. Each antibody was reacted at room temperature for 20 minutes, and then reacted with biotinylated secondary antibody and streptavidin-peroxidase, followed by reaction with DAB solution containing Hydrogen peroxide and Nickel Solution. After counter staining in fast red solution, dehydration and mounting were performed. Finally, it was observed through a Olympus BX41 microscope. Figure 13a is a result of immunostaining of H & E and mulit-cytokeratin, HMB45, collagen type-1, vimentin, E-cad after culturing the cells cultured by the skin cell co-culture method of the present invention. 13b is a photograph comparing normal skin and skin tissue formed in vivo according to the present invention. After co-culture of keratinocytes, fibroblasts and melanocytes, the animal test showed melanin distribution similar to normal skin as well as the formation of dermis and epidermis. Therefore, it can be seen that the cells co-cultured according to the present invention exhibit a skin regeneration effect very similar to that of normal skin.

In contrast, only Keratinocytes were cultured alone, or Keratinocytes and fibroblasts were cultured alone and then mixed. FIG. 14 shows the results of H & E and mulit-cytokeratin immunostaining after culturing only Keratinocytes alone. The epidermal layer was formed very weakly, and the epidermal layer formed by the multi-cytokeratine immunostaining was very thin and poorly located. FIG. 15 shows that Keratinocytes and fibroblasts were cultured alone and administered together in a 1: 1 ratio, and then H & E (A), human epidermal cell markers, plate-cytokeratin (B), cytokeratin 14 (C) Antibodies staining results of, vimentin (LA), collagen type IV (MA) respectively. Compared with the result of epidermal cells alone, formation of basement membrane (collagen type IV) was induced and epidermal and dermal layers were formed, but skin color could not be expressed by the melanin cell.

As described above, according to the present invention, by mixing and culturing various skin cells isolated from one donor site, all cells grow well in a serum-free medium by paracrine effect between cells and culture each cell. In order to grow well in a medium other than the specialized medium to grow, it grows well even if the cell number is too small to grow when cultured alone, freezing and thawing melanin pigment cells was almost impossible in single culture, but thawing in the case of mixed co-culture and freezing It grows well afterwards, and the expression of smooth muscle actin (SMA), which is derived from in vitro fibroblast cultures, is not induced in mixed co-cultures (i.e., culture of fibroblasts without differentiation is possible). Cell therapy comprising the mixed cultured skin cells, only epidermal cells (Keratinocyte) It showed a better skin healing effect than the cell therapy.

1 is a photograph observing the cell shape in the primary culture (P = 1) and subculture (P = 2 to 5) mixed culture in accordance with the present invention.

2 is a photograph comparing the case of mixed culture and the case of culturing fibroblasts and epidermal cells alone according to the present invention.

Figure 3 is a test result by varying the mixing ratio of the cells in the mixed culture according to the present invention.

FIG. 4 is a photograph comparing the culture of epidermal cells alone and the culture of epidermal cells and fibroblasts in a medium to which serum is added and a medium without serum.

5 is a photograph confirming cell viability and cell state after cryopreservation and thawing of cells cultured according to the present invention.

Figure 6 is a photograph of immunofluorescence staining of co-cultured cells of passage number 2 according to the present invention.

Figure 7 is an immunofluorescence stained picture of passage co-culture of passage number 2 according to the present invention.

8 is an immunofluorescence stained image of passage co-cultured cells of passage 3 according to the present invention.

9 is a photograph of immunofluorescence staining of cells co-cultured with passage number 3 according to the present invention.

10 is an immunofluorescence stained picture of passage co-cultured cells of passage 4 according to the present invention.

11 is a photograph confirming that α-SMA (α-smooth muscle actin) is expressed in mixed co-cultured cells according to the present invention.

Figure 12 is a photograph of in vivo skin formation ability in nude mice of mixed co-cultured cells according to the present invention.

13 is a result of immunostaining of H & E and mulit-cytokeratin, HMB45, collagen type-1, vimentin, and E-cad after culturing the cells cultured by the skin cell co-culture method of the present invention.

FIG. 14 shows the results of H & E and mulit-cytokeratin immunostaining after culturing only epidermal cells (Keratinocyte) alone.

Fig. 15 shows that epidermal cells and fibroblasts alone are cultured and mixed, and then, after animal testing, H & E (A), human epidermal cell markers, plate-cytokeratin (B), cytokeratin 14 (C), and bimentin (L). ), Collagen type IV (e) antibody staining results of each.

Claims (8)

Epidermal cells (Keratinocytes), melanocytes (Melanocytes), Fibroblasts and Endothelial cells isolated from one donor site derived from autologous skin tissue were serum-free medium ( Method for co-culture of skin cells, characterized in that the mixed culture by the paracrine effect between the cells without supplying the cytokine (cytokine) or growth factor (growth factor) required for the sole culture in serum-free media. The method of claim 1, wherein the size of the one donor site is a skin cell co-culture method, characterized in that 0.5-2cm ² . The method of claim 1, wherein the epidermal cells (Keratinocyte) and melanocytes (Melanocyte) are separated from the epidermal layer of the donor site, the fibroblast and endothelial cells (endothelial cells) are separated from the dermal layer of the donor site Skin cell co-culture method characterized in that. delete The method of claim 1, wherein the ratio of epidermal cells + melanocytes: fibroblasts + vascular endothelial cells is cultured in a mixed culture of 15: 1 to 25: 1. The method of claim 1, wherein the expression of smooth muscle actin (SMA) induced in the culture of fibroblasts alone is not induced. delete Including epidermal cells (Keratinocyte), melanocytes (Melanocyte), Fibroblast and Endothelial cells co-cultured according to any one of claims 1, 2, 3, 5, or 6 Wound healing cell therapeutic composition.

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