KR101922346B1 - Mesenchymal stem cells spheroid-sheet complex for treating ulcer and preparation method thereof - Google Patents

Abstract

The present invention relates to a stem cell spheroid-sheet complex and a preparation method thereof. The preparation method of the stem cell spheroid-sheet complex comprises: a step (A) of preparing a temperature-sensitive hydrogel; a step (B) of culturing a stem cell with a single cell form in a three-dimensional culture dish to prepare a stem cell spheroid; a step (C) of dividing the stem cell spheroid in the hydrogel to culture the stem cell spheroid; and a step (D) of cooling the surface of the hydrogel to separate the stem cell spheroid that has been cultured on the surface of the hydrogel in the form of a sheet. More specifically, the stem cell spheroid-sheet complex of the present invention can treat the lesion by expressing the bioactive factors which are not separated but remain to be required in tissue regeneration when bioactive factors are applied to a lesion exposed to the outside. The stem cell spheroid-sheet complex improves tissue regeneration capability of stem cells by having a high expression ratio of the bioactive factors and has an advantage of a significant ulcer treatment effect accordingly. Further, the preparation method of the present invention can minimize loss of the stem cells according to injection of the stem cells by injecting the temperature-sensitive hydrogel under a conventional lesion tissue with a needle. Therefore, the present invention is expected to be used as a new concept cell treatment technology for inducing regeneration of various damaged tissues of Pharyngeal fistula, tracheostenosis, cornea deficit, skin deficit, stomach ulcer, oral ulcer or the like, and treating the lesions exposed to the outside.

Description

[0001] The present invention relates to a mesenchymal stem cell spheroid-sheet complex for treating ulcer and a method for preparing the same,

The present invention relates to a stem cell sponge-sheet complex for the treatment of ulcers, and more particularly to a stem cell sponge-sheet complex for the treatment of ulcers, and more particularly, And a method for producing the same.

The oral cavity is composed of mucous membranes. The mucosa acts as a primary defense against external homeostasis, external infections, and acts as a channel for nutrients and drugs to be absorbed. If the mucosa is defective, it causes bacterial infection and loss of organ function due to drying of submucosal tissue. In the past, the most important side effects of chemotherapy were vomiting and reduction of immunity due to myelosuppression. However, it has been solved by the rapid development of antiepileptic drugs and hematopoietic growth factors. Recently, however, mucositis has become the most serious side effect have. In addition, infection caused by oral mucositis may cause death due to systemic sepsis in immunocompromised state by chemotherapy.

Intractable oral ulcers lead to pain and decreased nutritional intake, increased need for parenteral nutrition, and reduced patient life. In cancer patients with neutropenia, 10% of patients with severe mucositis are still at high risk for sepsis. In addition, planned chemotherapy and radiotherapy dose reduction, treatment interruption, inhibition of simultaneous treatment with chemotherapy and radiation, and subsequent cancer recurrence are increasing. In addition, the onset of oral mucositis requires additional treatments and procedures such as narcotic analgesics to control pain, antibiotics to prevent infection, parenteral nutrition, and gastrointestinal cirrhosis. About twice as much. The number of domestic oral mucositis patients increased by about 30% for 6 years, and the payment of medical expenses rose by about 70% (2008 ~ 2014, Health Chosun).

Nevertheless, there is currently no direct treatment for oral ulcers (mucositis). In the case of treatment using growth factors, oral cancer was not induced and FDA approval was not obtained. Also, treatment using anti-inflammatory drugs was not approved by the FDA in clinical trials.

On the other hand, various attempts have been made to use stem cells as therapeutic agents for tissue regeneration. However, when stem cells are present in the form of single cells, they are not attached to tissues when applied to tissues. There is a problem that can not be applied to a tissue (open tissue) exposed to the outside of the tissue. In the case of a tissue culture dish which is easy to adhere to cells, considering that at least 6 to 12 hours are required for cell attachment, when a single cell type stem cell is introduced into an open tissue, There was a problem. Accordingly, there has been a need for research on the development of an effective stem cell therapeutic agent for treating open tissue (for example, oral mucosa, etc.).

Journal of Oral and Maxillofacial Surgery, Vol. 28, No. 2, 2002.4, 147-154, "Immunohistochemical Study on the Vascular Endothelial Growth Factor (VEGF) Expression in Genistein-

The inventors of the present invention have conducted intensive studies on the treatment of intractable severe oral ulcers using stem cells. As a result, they have found that the therapeutic effect of the present invention is excellent when the stem cell spoloids cultured in 3D spoloid form are applied to the affected part in the sheet form, Completed.

Accordingly, the present invention has an object to provide a stem cell sphere-sheet complex and a method for producing the same.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object, the present invention provides a method for producing a thermosensitive hygroscopic gel, comprising the steps of: (A) (B) culturing a single cell type of stem cells in a 3D culture dish to produce a stem cell spoloid; (C) dividing and culturing the stem cell spleod into the hydrogel; And (D) cooling the surface of the hydrogel to separate the stem cell sprooid cultured on the surface of the hydrogel into a sheet form. The present invention also provides a method for producing a stem cell sprooid-sheet complex.

In one embodiment of the present invention, the stem cell spleoid-sheet complex may be for treating a opened tissue defect.

In another embodiment of the present invention, the temperature-sensitive hydrogel of the step (A) may include a cell adhesion protein.

In another embodiment of the present invention, the cell adhesion protein may be fibronectin.

In another embodiment of the present invention, the step (C) may be performed by dividing the stem cell spleod into 4 to 400 per unit area (cm 2) of the hydrated gel in the hydrated gel.

In another embodiment of the present invention, the step (D) may be performed by cooling the surface of the hydrated gel to 0 ° C to 25 ° C to separate the stem cell sprooid cultured on the hydrogel surface into a sheet form.

The present invention also provides a stem cell spleen-sheet complex produced by the above-described method, wherein the complex may be for treating an open tissue defect.

The present invention also provides a method for treating an open tissue defect comprising the step of transferring the stem cell spleen-sheet complex to an open tissue of an individual's defect.

The present invention also provides a process for preparing a thermosensitive hydrogel comprising: (a) preparing a thermosensitive hydrogel; (b) culturing single cell type stem cells in a 3D culture dish to prepare stem cell spheroids; (c) dividing the stem cell spleen into the hydrogel and culturing it in the form of a sheet; (d) moving the hydrogel to the affected area so that the stem cell culture surface contacts the affected part; And (e) cooling the surface of the hydrated gel to separate the stem cell sprooid cultured on the surface of the hydrogel into a sheet form and removing the hydrogel.

The present invention also relates to a method for preparing a thermosensitive hydrogel comprising the steps of: (a) preparing a thermosensitive hydrogel; (B) dividing and culturing the stem cells on the surface of the hydrated gel; (C) cooling the surface of the hydrogel to separate the stem cells cultured on the hydrogel surface into a sheet form; And (d) moving the separated stem cell sheet to the affected part of the individual.

In one embodiment of the present invention, the temperature-sensitive hydrogel of the step (a) may include a cell adhesion protein.

In another embodiment of the present invention, the cell adhesion protein may be fibronectin.

In addition, the present invention provides a use for treating an open tissue defect of the stem cell sheet.

In addition, the present invention provides the use of said stem cell spleen-sheet complex for the treatment of open tissue defect.

In one embodiment of the present invention, the open tissue defect may be at least one selected from the group consisting of pharyngeal fistula, tracheal stenosis, corneal defect, skin defect, gastric ulcer, and oral ulcer.

The stem cell spolide-sheet complex according to the present invention can treat a lesion by releasing bioactivity factors required for tissue regeneration without remaining for a long time when applied to a lesion exposed to the outside, unlike a single cell stem cell, The stem cell spoloid-sheet complex has a significantly higher expression level of a bioactive factor than a stem cell sheet, thereby improving the tissue regeneration ability of the stem cell and thus having a significant therapeutic effect on the ulcer.

In addition, in the method for preparing a stem cell spleen-sheet complex according to the present invention, stem cells can be minimized by injecting stem cells with a needle under the conventional lesion tissue using a thermosensitive hydrogel, By applying the cell adhesion protein to the sensitive hydrogel, the survival rate of stem cells can be maintained high.

Accordingly, the present invention can be used as a new concept cell therapy technology for inducing regeneration of various injured tissues such as pharyngeal fistula, tracheal stenosis, corneal defect, skin defect, stomach ulcer, oral ulcer, and lesions exposed to the outside do.

1 is a schematic diagram illustrating application of a stem cell sheet or a stem cell spheroid-sheet complex according to the present invention to oral ulcer treatment.
Figure 2 is a photograph of an animal model of severe oral ulcer.
FIG. 3 is a view showing a change in diameter of the temperature-sensitive hydrogel according to temperature.
FIG. 4 is a graph showing the changes in VEGF and MMP-1 levels of adipose derived stem cells, bone marrow-derived stem cells, and pluripotent stem cells derived from adipose-derived stem cells cultured in sheet form (2D) The amount of protein expression is compared and confirmed.
FIG. 5A is a view showing a state in which a stem cell sheet on the surface of a hydrogel is delivered to a culture dish and a stem cell sheet delivery efficiency over time, and FIG. 5B is a view showing a survival rate of a stem cell delivered to a culture dish.
FIG. 6A is a diagram showing stem cell spoloids prepared in a 3D culture dish, and FIG. 6B is a diagram showing a stem cell spoloid-sheet complex cultured on a temperature-sensitive hydrogel containing a cell adhesion protein for 24 hours.
FIG. 7 is a graph showing the amount of cells remaining on the surface of a thermosensitive hydrogel before and after transferring a stem cell sheet or a stem cell spheroid-sheet complex to an oral ulcer lesion.
FIG. 8 is a view showing that stem cells transferred to oral ulcer lesions using a stem cell sheet remain in lesions applied for 7 days.
9 is a comparative evaluation of the effectiveness of AD-MSC sheet or spleed-sheet complex for the treatment of severe oral ulcers.

The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated and described in the drawings. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

In the treatment of open tissues (open tissues) exposed to the skin such as skin using stem cells, stem cells must be able to adhere to the lesion for as long as possible to survive, In the case of treating a stem cell with a lesion, excessive cells are lost, the stem cell easily disappeared from the lesion, and the stem cell adhered to the lesion is easily killed.

Thus, the present inventors confirmed that, unlike stem cell single cells, the stem cell sheet adheres to the site of the open tissue for a long time without being disappeared (see Examples 5 and 8) In the case of the cell sphere-sheet complex, the amount of bioactive factor required for tissue regeneration is increased by about 5 to 10 times as much as that of the stem cell sheet, (See Examples 4 and 7). In addition, the use of a thermo-sensitive hydrogel containing a cell adhesion protein in the preparation of a stem cell sheet and a stem cell sphere-sheet complex reduces the loss of stem cells caused by application to lesions, And the survival rate can be maintained at a high level.

Accordingly, the present invention provides a method for producing a thermosensitive hygroscopic gel, comprising the steps of: (A) (B) culturing a single cell type of stem cells in a 3D culture dish to produce a stem cell spoloid; (C) dividing and culturing the stem cell spleod into the hydrogel; And (D) cooling the surface of the hydrogel to separate the stem cell sprooid cultured on the surface of the hydrogel into a sheet form. The present invention also provides a method for producing a stem cell sprooid-sheet complex.

According to an aspect of the present invention, the stem cell spleen-sheet complex may be used for treating an open tissue defect, preferably for treatment of skin damage and / or ulcer, and the ulcer includes peptic ulcer, corneal ulcer (Ulcer), skin ulcer (ulcer ulcer), diabetic ulcer, arteriosclerotic ulcer, congestive ulcer, syphilis ulcer, ductal ulcer, herpetic ulcer (genitourinary ulcer), aphtha stomatitis, Behcet's disease, oral ulcer The peptic ulcer may include Barrett's ulcer, Curling ulcer, Cushing ulcer, plant ulcer, colonic ulcer, and the like. Also, non-limiting examples of the open defect include pharyngeal fistula, tracheal stenosis, corneal defect, skin defect, gastric ulcer, and oral ulcer.

The oral mucosa is a structure in which keratinization is not relatively performed. In particular, the oral cavity frequently moves. In the treatment of stem cells, the oocyte is often dislodged when treated with a single cell. However, Can effectively adhere to the mucosa and remain for a long time.

In addition, since the oral mucosa is rich in blood vessels, bioactive factors secreted from stem cells applied to oral lesions can actively act. On the other hand, it has been confirmed that the expression level of bioactive factors is higher than that of the spleen-like stem cells in the sheet form, and the spleen form and the sheet form are combined to form a stem cell spleed-sheet complex, It is possible to dramatically increase the therapeutic effect by increasing the adhesion to the oral mucosa while expressing a large amount of bioactive factors.

According to another aspect of the present invention, the temperature-sensitive hydrogel of step (A) may include a cell adhesion molecule (CAM). In the present invention, the term "cell adhesion protein" matrix, which facilitates the binding of cells to other substances, such as, for example, Cadherin, fibrinogen, fibronectin, vitroectin, laminin, collagen, , Selectin, etc., but may be preferably fibronectin.

According to another aspect of the present invention, in step (C), the number of stem cell spleoids dispensed in the hydrogel may vary depending on the size of the lesion, the size of the hydrogel, and the like. The size of the hydrogel is determined by the size of the lesion to which the stem cell spolide-sheet complex is to be applied, and the stem cell spolide-sheet complex is appropriately adhered without detaching from the lesion, Size or similar. In the case of applying the stem cell spoloid-sheet complex provided by the present invention to the oral mucosa, the stem cell spoloid-sheet complex must be small in size and flexible because the mouth is highly motion and its movement is delicate.

Considering that the number of stem cell spoloids distributed in the hydrated gel varies depending on the size of the lesion, but growth factors act as self-inhibitors at high concentrations, high concentrations of bioactivity factors expressed from a large amount of spoloids There is a possibility that the tissue regeneration can be suppressed. Further, considering that the bioactive factors must maintain a therapeutic concentration for tissue regeneration, there is a problem that the bioactive factors expressed in a small amount of the sphere do not play a sufficient role. Therefore, the number of stem cell spheroids to be dispensed in the hydrated gel in the step (C) may be variable, but it is preferable that the number of the stem cell spheroids distributed in the hydrated gel is 4 or more per unit area (cm 2) 400, and more preferably 40 to 110,

The thermosensitive hydrogel used in the present invention is composed of a thermosensitive polymer exhibiting a lower critical solution temperature (LCST), and the degree of freedom of the polymer is lowered as the temperature is increased. That is, it has a property of shrinking at a temperature higher than LCST and expanding at a lower temperature. When a hydrogel having a stem cell sheet or a stem cell sphere-sheet composite on its surface is attached to the oral mucosa using the above-mentioned characteristics of the thermosensitive hydrogel, the hydrogel does not expand the volume of the hydrogel, After a sufficient time has elapsed, the hydrated gel is cooled by cooling the surface of the hydrogel to separate the hydrogel and the stem cell sheet or the stem cell spheroid-sheet complex, and without loss of stem cells and / or damage Can be delivered to the lesion.

Thus, according to an aspect of the present invention, the step (D) may be a step of cooling the surface of the hydrogel to 0 ° C to 10 ° C to separate the stem cell sprooid cultured on the surface of the hydrogel into a sheet form , The cooling temperature may preferably be 0 캜 to 25 캜, more preferably 4 캜 to 10 캜. If the cooling temperature is higher than 25 ° C, the hydration gel may not be separated from the stem cell sheet or the stem cell sphere-sheet complex due to a low expansion ratio of the hydrogel, and when the temperature is lower than 0 ° C, Segregation between cell spoloid-sheet complexes is smooth, but it may cause stem cell loss due to cell death of stem cells.

The delivery time of the stem cell sheet or stem cell sphere-sheet complex is proportional to the cooling temperature.

The present invention also provides a method for treating an open tissue defect comprising the step of transferring the stem cell spleen-sheet complex to the affected part of the individual. In the present invention, " opened tissue " means a tissue exposed to the external environment including skin. In the present invention, " open tissue defect " means a tissue defect in the open tissue Means that the cells are lost, and includes skin damage and ulcers.

In the present invention, the term " ulcer " means that the epithelium is removed due to inflammation, necrosis, etc., and the surface of the tissue is locally deficient or depressed. The ulcer refers to peptic ulcer, corneal ulcer (eye ulcer), skin ulcer , A diabetic ulcer, an arteriosclerotic ulcer, a congestive ulcer, a ulcer due to syphilis, a soft laxity, a herpetic ulcer (genitourinary ulcer), an aphthae stomatitis, a Behcet's disease, an oral ulcer, have. In the present invention, open tissue defect treatment is interpreted as including the treatment of ulcer, and the open tissue and ulcer requiring the treatment may be mixed.

The term " individual " as used herein refers to a subject in need of treatment of a disease, and more particularly, a human or non-human primate, mouse, rat, dog, cat, horse, rabbit, And mammals such as cows.

The present invention also relates to a method for preparing a thermosensitive hydrogel comprising the steps of: (a) preparing a thermosensitive hydrogel; (B) dividing and culturing the stem cells on the surface of the hydrated gel; (C) cooling the surface of the hydrogel to separate the stem cells cultured on the hydrogel surface into a sheet form; And (d) moving the separated stem cell sheet to the affected part of the individual.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1. Severe oral ulcer animal model

Rabbit severe oral ulcer animal models were induced. As shown in Fig. 2, the oral ulcer animal model was prepared by causing a filter paper having a diameter of about 4 mm impregnated with 70% acetic acid (200 μl) in a rabbit gingival oral mucosa for 1 minute to produce a ulcer having a diameter of about 6 mm . The rabbits used in the oral ulcer animal model were male New Zealand white rabbits and maintained a body weight of 3.0 to 3.2 kg at the time of the procedure.

In the following experiments, the application of the stem cell sheet or stem cell sphere-sheet composite was performed 3 days after induction of oral ulcer.

Example  2. Temperature Sensitivity Hydrated gel  Produce

Thermosensitive hydrogel polymer Tetronic ^® 1307 (Tetronic-tyramine; Tet-TA ) a functional terminal group substituted with tyramine for the production of a was used. The powdered Tet-TA polymer was dissolved in phosphate buffered saline (PBS) at a concentration of 12% (w / v), added with fibronectin (final concentration, 50 μg / Was prepared by dividing the solution. The first tube contained H ₂ O ₂ (HRP, 0.0025 mg / ml) solution was added to the second tube. After mixing each solution thoroughly, the two prepared solutions were mixed at a ratio of 1: 1, Were injected between glass plates spaced with Teflon spacer (0.5 mm). The mixed solution reacted between the glass plates to form a thermosensitive hydrated gel, which was separated from the glass plate after about 10 minutes of reaction. The separated hydrogels were cut into 8 ㎜ diameter disks using biopsy punch. After sterilization, they were exposed to UV for 30 minutes and stored in a 37 ℃ incubator until they were used.

Next, to evaluate the rate of expansion of the thermosensitive hydrogel, the size of the hydrated gel stored at 37 ° C and 4 ° C for 1 day was measured, and the diameter of the hydrogel was quantitatively analyzed.

As a result, as shown in FIG. 3, it was confirmed that the diameter of the hydrogel prepared was about 1.4 times as large as that of the hydrated gel when it was changed from 37 ° C to 4 ° C.

Example  3. Stem cells Of Spheroid  Produce

For the production of stem cell spleens, adipose-derived mesenchymal stem cells (AD-MSC) were isolated from tissue culture dishes by trypsin / EDTA treatment and separated into single cells. Considering the number of single cells obtained, the cell culture medium was added so that the concentration of the single cell solution was 1 × 10 ⁶ cells / ml. 1 ml of the cell solution was cultured in a 3D culture dish (StemFit ^® 3D, Microfit, Korea) for 3 days.

Example  4. Stem cells Spearoid  Confirmation of tissue regeneration effective ingredient expression

The expression level of the effective protein involved in regeneration of stem cell spoloid was confirmed. Specifically, the amount of expression of vascular epithelial growth factor (VEGF), an effective protein involved in neovascularization, and the expression level of matrix metalloproteinase-1 (MMP-1), an effective protein involved in cell migration, were quantitated by ELISA. The stem cells are derived from adipose-derived mesenchymal stem cells (AD-MSC), bone marrow-derived mesenchymal stem cells (BM-MSC), induced pluripotent stem cells -derived mesenchymal stem cells (iPS-MSC) were each cultured in the form of spheroids (3D) by the method of Example 3 above. As a control, AD-MSC was cultured in sheet form (2D) to quantitate VEGF and MMP-1 expression.

As a result, in the case of VEGF expression amount, as shown in the left panel of FIG. 4, a high expression level was confirmed in AD-MSC spoil and BM-MSC speoldo, and about three times as high as in iPS-MSC speold Respectively. In addition, AD-MSC spolide showed approximately 16-fold higher expression of VEGF than AD-MSC sheet.

In the case of MMP-1 expression, as shown in the right panel of FIG. 4, the expression levels of AD-MSC sphaeroide were about 3-fold and 5-fold higher than that of BM-MSC and iPS- Respectively. In addition, AD-MSC spleod compared to AD-MSC sheet showed about 7-fold higher expression of MMP-1.

From the above, it was confirmed that the expression levels of tissue regeneration effective ingredients were increased in the stem cells cultured in the spoloid form as compared with the sheet-type stem cells, and the expression amount of the regenerating active ingredient of AD-MSC spolod was the most Respectively.

Example  5. Hydrating Gel  Production of stem cell sheets from the surface

AD-MSC was used for the production of stem cell sheets. AD-MSC was prepared by using MesenPro RS kit (Gibco), and then cultured in a tissue culture dish at 37 ° C and 5% CO ₂ to ensure sufficient cell numbers. The cultured cells were washed twice with PBS and treated with trypsin / EDTA for 3 minutes to obtain single cells. Then, the cells were incubated with a temperature sensitive hydrated gel Lt; ⁵ > cells / cm < ^{2 &} gt ;. After 24 hours of cell division, a single layer of stem cell sheet was formed on the hydrogel surface.

Next, in order to examine the ability of the hydrogel to transmit the stem cell sheet, the hydrogel formed with the stem cell sheet was turned upside down on the culture dish to induce adhesion between the cells and the bottom surface at 37 ° C for 15 minutes, For 5 minutes, 10 minutes, and 15 minutes, respectively, to induce the natural elimination of the stem cell sheet and remove the hydrogel from the hydrogel in the culture dish. After passing the stem cell sheet in the culture dish by the above method, in order to determine the survival rate of cells was conducted to Live / dead assay (LIVE / DEAD ® Viability / Cytotoxicity Kit for mammalian cells, ThermoFisher), as a result the living cells are green ( Calcein-AM) cells were stained with red (ethidium homodimer-1), which was confirmed by fluorescence microscopy (NIKON epifluorescence microscope).

As a result, as shown in FIG. 5A, it was confirmed that the cell sheet delivery efficiency was as high as 80% or more in the hydrogel which was expanded for 10 minutes. In addition, as shown in FIG. 5B, it was confirmed that the thus delivered stem cells had a high survival rate of about 90%.

Example  6. Stem cells Speroid - Preparation of sheet composite

17-20 stem cell spleens prepared by the method of Example 3 were dispensed on the temperature sensitive hydrogel in which the cell adhesion protein prepared in Example 2 was introduced and then cultured for 24 hours. Lid-sheet composite. At this time, the total number of cells constituting 17-20 stem cell spoloids was made equal to the total number of cells constituting the stem cell sheet (2D). FIG. 6A shows the shape of stem cell spoloids prepared by the method of Example 3, FIG. 6B shows the shape of the stem cell spoloid-sheet complex prepared by Example 6, and the complex is applied to oral ulcer lesions Respectively.

Example  7. Oral ulceration lesions are stem cell sheets or stem cells Speroid - Evaluation of transfer efficiency of sheet composite

In order to confirm the delivery efficiency of the stem cell sheet and the stem cell spoloid-sheet complex prepared according to Examples 5 and 6 to oral ulcer lesions, only the stem cells were treated with a fluorescent material (Vybrant ^® DiD Cell-labeling Solution, Invitrogen) , A stem cell sheet and a stem cell spleed-sheet complex were prepared according to Examples 5 and 6 above. The hydrogel gel was then applied to the surface of the hydrogel so that the cells could reach the lesion of the oral ulcer by the stem cell sheet or the stem cell sphere - sheet complex. After 15 minutes, the hydrogel was inflated to deliver the stem cells into the lesion, and PBS (4 ° C) was flowed over the hydrogel for 10 minutes. To confirm whether stem cells were transferred from the hydrogel, we observed the remaining cells on the hydrogel before and after stem cell transfection through a fluorescence microscope.

As a result, as shown in Fig. 7, it was confirmed that the stem cell sheet or the stem cell sphere-sheet complex on the hydrogel was all transferred to the lesion site.

Example  8. Assessment of residual stem cells in oral ulcer lesions

In order to confirm the remnant of the stem cells delivered from the hydrogel, the stem cell sheet tagged with a fluorescent material (Vybrant ^® DiD Cell-labeling Solution, Invitrogen) was transferred to the oral ulcer, and the IVIS (in vivo imaging system) The results are shown in Fig. As a control group, a hydrogel without a stem cell sheet was applied to oral ulcer lesions. After 1, 4, and 7 days of stem cell sheet transfer, oral ulcer lesions were harvested and observed with IVIS.

As a result, as shown in FIG. 8, the amount of stem cells delivered decreased with time, but it was confirmed that the stem cells remained in the lesion even 7 days after the delivery. The above results suggest that stem cells can be applied as an effective therapeutic agent even to open lesions.

Example  9. Stem cell sheet or stem cell Speroid - Evaluation of efficacy of oral complex for treating oral ulcers

AD-MSC, which was identified as the best stem cell in the amount of expression of tissue regeneration effective ingredient in Example 4, was used for the evaluation of oral ulcer treatment efficacy. The AD-MSC sheet or spleoid-sheet complex was applied to the oral ulcer lesions of the rabbits modeled in Example 1, and the size of the wounds at 4 and 7 days was quantitated. Specifically, the AD-MSC sheet or the spleen-sheet complex was applied to the ulcer lesions. On day 7, the regenerated mucosa was harvested, and keratin5 (K5) and keratin13 (CK13) were observed through H & E staining and immunohistochemistry Respectively.

As a result, as shown in FIG. 9, when the stem cell sheet or the stem cell spoloid-sheet complex was applied to the lesion of the oral ulcer, it was confirmed that there was no significant difference in the size change of the wound upon visual observation. However, when histopathologically examined, about 80% of the mucous membrane applied with stem cell sheet was closed and many immune cells gathered at the lesion site, whereas the mucosa applied with the stem cell spheroid-sheet complex had 100 % Were closed and no inflammatory cells were observed. In addition, the expression of K5 and CK13 was not observed in the mucous membrane to which the stem cell sheet was applied. On the other hand, in the mucous membrane regenerated by applying the stem cell spoloid-sheet complex, K5 expression, which is known as a protein expressing progenitor-like cells of the basal layer, was confirmed and CK13 expression, which is known as a protein expressed in the suprabasal layer, It was confirmed that the regenerated mucosa was rebuilt with basal-suprabasal structure.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (9)

(A) a step of preparing a temperature-sensitive hydrogel;
(B) culturing a single cell type of stem cells in a 3D culture dish to produce a stem cell spoloid;
(C) dividing and culturing the stem cell spleod into the hydrogel; And
(D) cooling the surface of the hydrated gel to separate the stem cell sprooid cultured on the hydrogel surface into a sheet form.
The method according to claim 1,
Wherein said stem cell spoloid-sheet complex is for the treatment of opened tissue defects.
3. The method of claim 2,
Wherein the opened tissue defect is at least one selected from the group consisting of pharyngeal fistula, tracheal stenosis, corneal defect, skin defect, stomach ulcer, and oral ulcer.
The method according to claim 1,
Wherein the temperature-sensitive hydrogel of the step (A) comprises a cell adhesion protein.
5. The method of claim 4,
Wherein the cell adhesion protein is fibronectin. ≪ RTI ID = 0.0 > 21. < / RTI >
The method according to claim 1,
Wherein the step (C) comprises culturing the stem cell sprooid in an amount of 4 to 400 per unit area (cm 2) of the hydrogel in the hydrogel, and culturing the stem cell sprooid .
The method according to claim 1,
Wherein the step (D) comprises cooling the surface of the hydrated gel to 0 ° C to 25 ° C to separate the stem cell sprooid cultured on the hydrogel surface into a sheet form. Gt;
8. A stem cell spleod-sheet complex, produced by the method of any one of claims 1 to 7, wherein said complex is for the treatment of a opened tissue defect, said stem cell sprooid- .
9. The method of claim 8,
Wherein the open tissue defect is at least one selected from the group consisting of pharyngeal fistula, tracheal stenosis, corneal defect, skin defect, gastric ulcer, and oral ulcer.

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