KR101328561B1 - Method for preparaing autologous agent for alleviation or treatment of skin defects - Google Patents

Abstract

The present invention relates to a method for producing an autologous skin cell therapy for alleviating or treating skin defects. Method for producing a cell therapy for alleviating or treating skin defects using autologous skin cells according to the present invention comprises the steps of obtaining normal skin tissue from the subject; Milling the obtained tissue into cells; It provides a method for producing a cell therapy comprising the step of obtaining a skin cell group comprising keratinocytes, melanocytes and fibrous cells from the tissue lysate after the grinding.
As a result, unlike the conventional method for preparing cell therapy, the enzyme treatment and / or culturing step are excluded, and thus, the preparation can be applied to the affected area by being prepared in a very simple and quick time.

Description

METHODS FOR PREPARAING AUTOLOGOUS AGENT FOR ALLEVIATION OR TREATMENT OF SKIN DEFECTS}

The present invention relates to a method for producing an autologous skin cell therapy for alleviating or treating skin defects. More specifically, unlike a conventional cell therapy, a cell therapy can be prepared in a short time by eliminating an enzyme or a culture step. Provide a way.

Defects in skin tissues such as burns, cuts, and depigmented skin can lead to scarring, prolonged periods of treatment, and other skin diseases if not properly treated or improperly treated.

In general, in the case of burns or wounds, conventionally, the skin was treated with gauze moistened with petrolatum-based ointment, and antibiotics such as penicillin and sulfamine were added to prevent secondary infection. In addition, a separate analgesic was administered to alleviate the pain. However, the long-term treatment of wounds for 2-3 weeks to 3 months is needed, and the cost is considerable.

In addition, in the case of burns, a method of treating using a cell therapy using autologous cells has been widely used. However, the conventional method for producing a cell therapeutic agent using autologous cells uses a method of harvesting specific cells by treating an enzyme and culturing the collected cells for several days. Therefore, the conventional cell therapy has a problem in that it is not suitable for a patient who takes a long time to incubate the cell and the degree of skin defect such as burn is serious and urgently needs treatment.

Therefore, an object of the present invention is a method for producing a cell therapy for alleviating or treating skin defects using autologous skin cells that are not isolated by enzymatic treatment, and for preparing cell therapy and cell therapy prepared by the above method. It is to provide a device.

The above object is, according to the present invention, a method for producing a cell therapy for alleviating or treating skin defects, comprising the steps of: obtaining normal skin tissue at one donor site derived from autologous skin tissue; Grinding the obtained normal skin tissue into cells; It can be achieved by providing a method for producing a cell therapy comprising the step of obtaining a skin cell group comprising at least any one of keratinocytes, melanocytes and fibrous cells from the tissue lysate after the grinding.

Here, the grinding step may be a step of grinding the normal skin tissue by cell units using a tissue mill for 10 to 60 seconds per 1 cm ² of the normal skin tissue size.

Here, the grinding step may be a step of grinding the normal skin tissue so that the concentration of the ground cells is 1 to 40 x 10 ⁷ / cm ² .

Here, the obtaining step, the obtained keratinocytes may be included in 60 to 90% of the total acquired cells.

Here, the obtaining step, the obtained melanocytes may be contained in 1 to 5% of the total obtained cells.

Here, in the obtaining step, the obtained fibrous cells may be included in 10 to 40% of the total obtained cells.

Here, the acquiring step may be characterized in that the skin cell group including the obtained keratinocytes, melanocytes and fibroblasts is not cultured.

Here, the acquiring step may be characterized in that it does not perform any enzyme treatment for acquiring a skin cell group including at least one of the keratinocytes, melanocytes and fibroblasts.

Here, the skin defect may include any one selected from the group consisting of skin wounds, wounds, skin scars, depigmented skin wounds due to burns.

In addition, according to the present invention, the above object can be achieved by a cell therapy prepared by the method for producing a cell therapy described above.

As described above, according to the present invention, as a method for producing a cell therapeutic agent for alleviating or treating skin defects using autologous skin cells, the cells separated from the cells by enzymatic treatment of autologous skin cells are also isolated. It can be prepared as a cell therapy without performing the culturing step, and has an effect that can be easily and quickly applied to the affected area. In addition, according to the present invention, even if the normal skin tissue obtained from the subject is a small amount, it has the effect of producing a cell therapy that can be applied to a wide affected area. In addition, there is an effect of providing a manufacturing apparatus capable of producing the cell therapy.

1 is a flow chart showing a method for producing a cell therapy according to an embodiment of the present invention,
2 is a diagram showing an example of a hemocytometer;
3a to 3b is a view showing the result of confirming the cell size of the cell therapy prepared according to an embodiment of the present invention,
Figure 4 is a view showing the clinical effect on the burn site of the cell therapy prepared according to an embodiment of the present invention,
5 is a view showing another clinical effect on the burn site of the cell therapy prepared according to an embodiment of the present invention,
Figure 6a is a view showing the electron microscope confirmation results of the cells isolated by the conventional enzyme treatment,
Figure 6b is a view showing the results of the electron microscope confirmation of the isolated cells according to an embodiment of the present invention,
7 is a view showing the effect of the animal experiment on the burn of the cell therapy prepared according to an embodiment of the present invention,
FIG. 8A is a view showing H & E staining results of the image part of FIG. 7;
FIG. 8B is a view illustrating MT staining results of the image part of FIG. 7;
9 is a view showing the effect of animal testing on the wound of the cell therapy prepared according to an embodiment of the present invention,
Figure 10a is a view showing the MT staining result after applying the control to the wound of Figure 9,
Figure 10b is a diagram showing the MT staining result after applying the cell therapy prepared according to an embodiment of the present invention to the wound site of Figure 9.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains. The present invention may be embodied in many different forms and is not limited to the embodiments described herein.

1 is a flowchart of a method for producing a cell therapy agent of the present invention.

As shown in Figure 1, the method for producing a cell therapy of the present invention comprises the steps of acquiring normal skin tissue at one donor site derived from autologous skin tissue (S11); Grinding the obtained normal skin tissue into cells (S12); And obtaining a skin cell group including at least one of keratinocytes, melanocytes, and fibrous cells from the tissue lysate after the grinding (S13).

Method for producing a cell therapy of the present invention is a method for producing a cell therapy for the relief or treatment of skin defects in mammals. Preferably, the present invention relates to a method for preparing a cell therapy agent for alleviating or treating skin defects in humans.

The acquiring step of the normal skin tissue may be obtained from the subject by using a skin extractor (Dermatome). The subject may include a mammal or a human. Preferably, to obtain normal skin tissue of a human having a skin defect on any part of the skin.

The normal skin tissue can be obtained by cutting to a size of (1 to 5mm) x (1 to 5mm), the number of tissue having the size can be obtained in consideration of the size of the affected area, for example the size It is possible to obtain 1 to 30 pieces, more preferably 5 to 25 pieces, and even more preferably 10 to 25 pieces.

The obtained normal skin tissue can be used by washing with physiological saline or annihilate.

In addition, the washed tissue can be used by sterilization using conventional methods.

The grinding of the normal skin tissue is a step of grinding the obtained normal skin tissue in units of cells. The milling can be carried out using a conventional tissue mill that can typically mill the tissue. When the tissue is pulverized, a small amount of physiological saline or sterile water may be pulverized in consideration of the amount of the tissue to be pulverized, and preferably 1-10 cc, more preferably 1-5 cc of physiological saline or sterile water is added. Can be pulverized.

When grinding the tissue using the tissue grinder, the grinding time can be set in consideration of the amount of the tissue, preferably 1 to 60 seconds per 1 cm ² of the tissue size, more preferably 10 to 60 The tissue can be milled cell-by-cell for seconds. The tissue may be pulverized so that the concentration of the pulverized cells is (1 to 40) x10 ⁷ cells / cm ² . The concentration of the cells may vary depending on the age of the subject or the condition of the tissue.

The acquiring of the skin cell group means acquiring a skin cell group including at least one of keratinocytes, melanocytes, and fibrous cells from the tissue lysate after the grinding.

The keratinocytes are keratinocytes that produce keratin and create physical barriers to protect individuals from harmful factors from the outside, and produce and secrete various biological response modulators that regulate immunological responses. It is an important component in regulating immune and inflammatory responses.

The melanocytes (melanocytes) are cells that produce melanin, a brown or black pigment present in animal tissues and skin, and continuously send melanin granules made in the cells to epidermal cells. The higher the amount, the more the skin color becomes yellowish brown to blackish brown, and the smaller the color becomes, the thinner the color becomes.

The fibroblasts, also called fibroblasts, are cells that form an important component of fibrous connective tissue and are flat and elongated when viewed with tissue sections and often have irregular projections.

Therefore, the acquiring step is to obtain a skin cell group including the keratinocytes, melanocytes, and fibrous cells from the tissue lysate after crushing the tissue.

At this time, the obtained skin cell group including at least one of keratinocytes, melanocytes, and fibrous cells may include not only cells that have survived but also cells that are destroyed in the grinding step.

Here, the obtained keratinocytes may be included in 60 to 90% of the total acquisition cells, the obtained melanocytes may be included in 1 to 5% of the total acquisition cells, the acquisition The fibroblasts can be included in 10 to 40% of the total acquired cells.

Thus, as described above, according to the present application, there is provided a method capable of separating keratinocytes, melanocytes, or fibrous cells from the normal skin tissue without treating any enzyme. In addition, there is provided a cell therapeutic agent which can be directly applied to the affected area without culturing a skin cell group including at least one of the obtained keratinocyte melanocytes and fibrous cells. Thereby, the cell therapy prepared by the manufacturing method according to the present invention is excluded from the cell separation process by the enzyme treatment, and the culture process of the separated cells is excluded, so that can be prepared in a very short time and immediately after the preparation to the affected area It is very effective to apply.

Here, the skin defect may include any one selected from the group consisting of skin wounds, wounds, skin scars, depigmented skin wounds due to burns.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

Experimental Example 1. Cell test and wound application examples contained in the cell therapy prepared according to an embodiment of the present invention

Normal skin tissues were obtained by cutting a skin tissue of 4x5mm size from a 47-year-old female and a 36-year-old female, each 20 using a skin extractor, washed with physiological saline, and sterilized. The sterilized tissue was pulverized for about 30 seconds using a tissue grinder with the addition of 2cc of sterile water.

When the grinding is completed, a 47-year-old female autologous skin cell was obtained by obtaining a skin cell group including at least one of keratinocytes, melanocytes, and fibrous cells from tissue lysates using a cell strainer. Cell therapy and cell therapy using autologous skin cells of 36 year old women were prepared.

1-1. Cell concentration measurement

Cell concentrations of the prepared 47-year-old women's autologous skin cells and 36-year-old women's autologous skin cells were measured using a hemocytometer.

3 shows an embodiment of a hemocytometer. As shown in FIG. 2, the hemocytometer has two chambers, which are divided into regular squares by slide glass with horizontal and vertical hatchings on the surface thereof, and microscopically examine cells existing in a constant volume rectangular section of each chamber. The number of cells can be counted under to determine the concentration of cells present in the entire sample.

100 μl of trypan blue was added to 100 μl of the prepared cell therapy product, mixed well, and slowly injected into a hemocytometer covered with a cover glass. After about 1 minute, the number of stained cells (dead cells) and non-stained cells (surviving cells) under the microscope was measured.

The concentration of the cells was measured by the following Equation 1.

[Equation 1]

(Sum of 1, 3, 7, 9 blocks)

As a result of the cell concentration measurement, the cell concentration of the 47-year-old woman using autologous skin cells was about 1.4x10 ⁸ cells per cm ^{2, and} the cell concentration of the 36-year-old woman using autologous skin cells was 2.4x10 ^8. Identified by cells.

1-2. Check cell size

The size of the keratinocytes, melanocytes, and fibroblasts of the 47-year-old female autologous skin cells and the 36-year-old autologous skin cells were determined. The procedure of the cell size confirmation experiment followed the general experimental procedure. As a comparative example, the size of primary cells, that is, normal cells, was confirmed. The cell size was confirmed under a microscope.

The result of the cell size experiment is as shown in Figures 3a to 3c.

FIG. 3a is a result of observing primary cells under a microscope as a comparative example, FIG. 3b is a result of observing a cell size of a cell therapy prepared using autologous skin cells of the 36-year-old woman, and FIG. 3c is The cell size of a 47-year-old woman using autologous skin cells was observed under a microscope.

As shown in Figure 3a, in the case of primary cells (comparative example) is the size of 10-25 μm, as shown in Figure 3b, the cells included in the cell therapy using autologous skin cells of the 36-year-old woman The size was determined to be 6-15 μm, and as shown in FIG. 3C, the size of the cells included in the cell therapy product using the autologous skin cells of the 47 year old female was also determined to be 6-15 μm. Therefore, it can be seen that the cell size included in each of the cellular therapeutic agents using autologous skin cells of the 47- and 36-year-old women is within the range of the size of normal cells.

1-3. Cell viability confirmation experiment

The survival rate of the cells contained in the 47-year-old female autologous skin cells and 36-year-old autologous skin cells were confirmed. 100 μl of each cell therapeutic agent 100 μl trypan blue was added and mixed well, and then slowly injected into a hemocytometer covered with a cover glass (see FIG. 2). After about 1 minute, the cell viability was calculated using the following equations (2) to (4) by measuring the number of cells (dead cells) and cells not dyed (surviving cells) that were stained under a microscope.

&Quot; (2) "

Number of living cells

(Sum of 1, 3, 7, 9 blocks)

&Quot; (3) "

Total cell count = Cell suspension (ml) X Cell concentration (cells / ml)

&Quot; (4) "

As a result of the experiment, the cell survival rate of the 47-year-old female cell therapy using autologous skin cells was 72%, 36-year-old female was 72.5%, showing a high cell viability.

1-4. Wound application

Cell therapy using autologous skin cells of the 47 year old woman was applied to the burn site of the 47 year old woman.

The burn affected tissue tissue adhesive (fibrin) first, and then sprayed and bandaged the cell therapy using the autologous skin cells. After applying the cell therapy, the state after 7 days was examined.

The results are as shown in FIG. As shown in Figure 4, when the cell therapy prepared by the manufacturing method of the present invention was sprayed on the burn site, it was confirmed that the cell regeneration of the burn site has a significant effect that can be confirmed with the naked eye.

Therefore, the method for producing a cell therapeutic agent of the present invention can be prepared without undergoing a step such as enzyme treatment and / or cell culture, and can be prepared in a fairly simple and fast time and immediately applied to the affected area, requiring urgent need. It can have a significant effect on the application of patients with skin defects.

Experimental Example 2 affected part 2 of the cell therapy prepared according to an embodiment of the present invention

Normal skin tissue from a 49-year-old male was obtained by cutting 20 pieces of skin tissue using a skin extractor, washed with physiological saline, and sterilized. The sterilized tissue was pulverized for about 30 seconds using a tissue grinder with the addition of 2cc of sterile water.

After completion of the grinding, by using a cell strainer (cell strainer) to obtain a skin cell group including at least any one of keratinocytes, melanocytes and fibroblasts from tissue lysate, autologous skin cells of 49-year-old male The cell therapy was used.

The 49-year-old male was first sprayed with a biotissue adhesive (fibrin), and sprayed and bandaged with a cell therapy including autologous skin cells. After applying the cell therapy, the state after 10 days, 30 days, and 155 days was examined.

The result of this application example is as shown in FIG. FIG. 5A is a photograph photographing a state immediately after an image of a 49-year-old male. 10 days after spraying the cell therapeutic agent containing the autologous skin cells to the burn site of (A) is in Figure 5 (B), 30 days after spraying is in Figure 5 (C), The state after 100 days after spraying is as shown in FIG. For this reason, it was confirmed that the skin cell regeneration of the burned site was well performed by only one spray of the cell therapeutic agent including autologous skin cells prepared according to the method of the present invention. As can be seen from the photograph of (D), the use of the cell therapy according to the present invention can be expected to have a preventive effect on the skin pigmentation abnormality often occurs due to burn pigment due to a small difference in pigmentation between the burn and normal skin. In addition, the boundary is very soft, and there is little scarring of graft scarring than tissue transplantation.

Experimental Example 3. Electron micrograph of the cells contained in the cell therapy

The cell therapy agent according to the production method of the present invention includes physically separated cells. Accordingly, the physically separated cells and the enzyme-separated cells were identified by electron microscopy.

Confirmation results are as shown in Figures 6a and 6b. Figure 6a is an electron micrograph of the cells separated by the enzyme, Figure 6b is an electron micrograph of the autogenous skin cells of 49-year-old male prepared in Experimental Example 2.

In general, cells are attached to each other by binding molecules. However, as shown in Figure 6a, when the cells are separated by the enzyme treatment, the enzyme will cut the cell adhesion molecules, so that the shape of each cell can be very clean.

Since the cells included in the cell therapy according to the present invention are not separated by enzymatic treatment but physically separated, it is confirmed that the cell adhesion molecule is not cut but is still attached to the separated cells as shown in FIG. 6B. Could. Therefore, when the cell therapy agent of the present invention is sprayed on the affected area, it is determined that the cell adhesion molecule is well attached to the affected tissue of the affected area to help skin regeneration.

Experimental Example 4. Animal Experiment

4-1. Burn site application example

Animal experiments were performed to confirm the effect on the burn site of the cell therapy prepared according to the present invention. A part of the skin of a 6 week old male nude was induced. In the same manner as in Experimental Example 1 or Experimental Example 2, a cell therapy including autologous skin cells was prepared using the normal skin tissue of the 6-week-old male nude rat and used as a test group. Saline was used as a control.

The control site and the test group were first applied to the burn site of the nude let, and the burn site was subjected to general dressing for 7 days, and the biopsy was performed at the expense of the nude hat after 14 days.

A tissue block using paraffin was prepared as the animal tissue of the burn site, and the tissue was cut into thin sections and attached to the slide to allow staining. The slides were observed by H & E (Haematoxylin and eosin stain) staining for observation of new tissues and cells, and then read and read. In addition, collagen staining was observed and read by MT stain (Masson's trichrome) staining method.

The results of performing the experiment are as shown in FIGS. 7 and 8.

Fig. 7 shows an example of application of the cell therapy agent of the present invention to a burn site of a nude rat.

As shown in Figure 7 (A) and (C) is a photograph showing the image portion of the nude let. (B) is a picture taken 14 days after spraying the control to the burn site of the nude let, (D) is a picture taken 14 days after spraying the experimental group on the burn site of the nude let.

In case of (B), the wound was still red, but in case of (D), the wound was not red but the same color as healthy skin in other areas.

Figure 8a is a view showing the results of the haematoxylin and eosin stain (H & E) staining. As shown in Figure 8a, (A) is a photograph obtained by performing normal skin cells H & E staining for comparison of the control group and the experimental group. (B) shows the results of H & E staining when the control group was applied to the burn site, and it was confirmed that the photographs of the normal cells and the test group were significantly different from the photograph (C) of the H & E staining results. That is, in the case of the control group (B), almost no epithelialization was made (arrow of B), and it was confirmed that the formation of skin appendages (glands, fat glands, hair follicles, etc.) was not performed at all. In contrast, in the case of the test group (C), the epithelialization was almost similar to that of normal tissues, and the formation of skin appendages was also formed to be almost similar to the normal cells.

8b is a diagram showing the results of MT stain (Masson's trichrome) staining.

As a result of performing MT staining to confirm the degree of collagen formation, it can be seen that in normal cells (A), the immature fibrosis layer was very thin, but the mature collagen (blue) occupies almost the majority. In the control group (B), the immature fibrosis layer (d) in the wound was considerably wide while the amount of mature collagen was extremely small.In the test group (C), the immature fibrosis layer (d ') was very thin and the amount of mature collagen was normal. It was confirmed that the formation is much similar to the cell.

As a result of the experiment, the test group to which the cell therapy of the present invention was applied as compared to the control group was found to be remarkably superior in epithelialization, skin appendages and regeneration of collagen.

4-2. Wound site application example

Animal experiments were performed to confirm the effect on the wound site of the cell therapy prepared according to the present invention. Induce wounds on a part of the skin of a 6-week-old male nude rat, and include autologous skin cells using the normal skin tissue of the 6-week-old male nude rat by the same method as in Experimental Example 1 or 2. Cell therapies were prepared and used as test groups. Saline was used as a control.

The control site and the test group were first sprayed onto the wound site of the nude let, and the wound site was subjected to a general dressing for 3 days, and the biopsy was performed at the expense of the nude let after 7 days. The wound was observed by staining collagen with MT satin staining, and the staining method was performed in the same manner as in the above Experiment 4-1.

9 is a view showing an application of the cell therapy of the present invention to the wound site of the nude rat.

As shown in Figure 9, (A) and (C) is a photograph showing the wound portion of the nude let. (B) is a picture taken 7 days after spraying the control to the wound site of the nude let, (D) is a picture taken 7 days after spraying the experimental group to the wound site of the nude let. In the case of the control group using saline, the epithelial cells were not regenerated at the wound site, but in the experimental group to which the cell therapy product of the present invention was applied for 7 days, the wound area was pale red. However, the epithelial cells have been shown to be regenerated, and the skin regeneration at the wound site was remarkably superior.

10A is a result of MT staining when the control group was applied to the wound site, immediately after the wound (A), 7 days after the control application (B), and 200-fold magnification (C) of the a site. As shown in Figure 9a, only in the control group using physiological saline, the formation of epithelialization at the wound site was low (B), very little of the MT stained collagen (C).

10b is a result of MT staining when the experimental group is applied to the wound site, immediately after the wound (A), 7 days after the experimental group is applied (B), and 200-fold magnification (C) of the b site.

As shown in Figure 10b, in the experimental group using the cell therapy according to the present invention, epithelialization was more active than the control group (B), it was confirmed that the amount of collagen (blue) is relatively large (C) . Therefore, the test group to which the cell therapy of the present invention was applied compared to the control group was found to be remarkably superior in epithelialization and collagen regeneration.

Although several embodiments of the present invention have been shown and described, those skilled in the art will appreciate that various modifications may be made without departing from the principles and spirit of the invention . The scope of the invention will be determined by the appended claims and their equivalents.

Claims (10)

As a method of manufacturing a cell therapy for alleviating or treating skin defects,
Grinding normal skin tissue derived from autologous skin tissue into cells;
Method for producing a cell therapy comprising the step of obtaining a skin cell group comprising at least one of keratinocytes, melanocytes, and fibrous cells from the tissue lysate after the grinding. The method of claim 1,
The grinding step, the method of producing a cell therapy, characterized in that for crushing the normal skin tissue in units of cells using a tissue grinder for 10 to 60 seconds per 1 cm ² of the normal skin tissue size. The method of claim 1,
The grinding step, the method of producing a cell therapy, characterized in that to crush the normal skin tissue so that the concentration of the pulverized cells 1 to 40x10 ⁷ / cm ² . The method of claim 1,
In the acquiring step, the obtained keratinocytes comprise 60 to 90% of the total acquired cells. The method of claim 1,
The acquiring step, wherein the obtained melanocytes cells are prepared from 1 to 5% of the total acquired cells. The method of claim 1,
In the acquiring step, the obtained fibrous cells comprise 10 to 40% of the total acquired cells. The method of claim 1,
The acquiring step, the method for producing a cell therapeutic agent, characterized in that the cultured skin cell group comprising at least one of the obtained keratinocyte melanocytes and fibrous cells. The method of claim 1,
The obtaining step, the method of producing a cell therapy, characterized in that the enzyme treatment is not performed to obtain a skin cell group comprising at least one of the keratinocytes, melanocytes and fibroblasts.
The method of claim 1,
The skin defect is any one selected from the group consisting of burns, wounds, skin scars, depigmented skin wounds. A cell therapeutic agent prepared according to the method of claim 1.

Images