KR20150072088A - 3D cell implant patch and process for preparing the same - Google Patents

Abstract

The present invention relates to a three-dimensional cell transplantation patch having cultured cells coupled to the fibrin gel coupled with heparin, a preparation method thereof, and a wound treating kit including the same. According to the present invention, the three-dimensional cell transplantation patch is formed to have the identical shape and tissue of a wounded area to be effectively used to treat the wound.

Description

TECHNICAL FIELD [0001] The present invention relates to a 3D cell implant patch and a manufacturing method thereof,

The present invention relates to a three-dimensional cell-freezing patch and a method for producing the same, and more particularly, to a three-dimensional cell-freezing patch in which cells cultured in a heparin-bonded fibrin gel are bound, a preparation method thereof, To a wound treatment kit comprising an edible patch.

Biomolecular engineering is a field of research that focuses on the repair of damaged tissues and organs in the event of injury to living tissues or organs due to disease or accident. In this regard, a method using a gel that can be injected as a supporter for regenerating a living tissue has been extensively studied. The method using the injectable gel can be easily injected into a necessary site without surgical procedures, , Cost, and so on. As the injectable gel, collagen gel, fibrin gel, alginic acid gel and the like have been used so far. In particular, since fibrin gel can be obtained from the patient's own blood, problems such as immunity induction do not occur, and thus it has attracted much attention as a support for biologic tissue engineering for autologous use.

Fibrin is a kind of light protein that is an insoluble protein caused by the action of enzyme thrombin in the plasma of fibrinogen. That is, fibrin forms a gel by enzymatic polymerization of fibrinogen in the presence of thrombin at room temperature. The fibrin gel thus formed is of great importance in the field of tissue engineering focusing on restoration of damaged tissues and organs. In particular, since the delivery of growth factor proteins is facilitated, fibrin gels in the field of tissue regeneration such as bones, Many studies related to gel have been carried out. Also, heparin, a type of glycosaminoglycan, has been used as an affinity ligand which binds to a certain class of heparin-binding proteins due to its highly negative charge property, which contains a large amount of sulfate groups. Such properties are mainly used for chromatographic separation in protein separation, but recently, attention has been paid to the possibility of applying the heparin-binding protein to a specific site in a body by immobilizing it on a carrier of a protein preparation. For example, WO 2005/028638 discloses a composition for coating the surface of a support containing fibrinogen and thrombin with fibrin, a support for cell sheet preparation whose surface is coated with fibrin, and a method for producing a cell sheet using the support Even though the cell sheet prepared by culturing the cells on the supporter has a multilayer laminate structure in the form of a sheet on which the cell layer is located, the binding between the cultured cells is very weak. Therefore, , There is a disadvantage in that the rate of engraftment in the living body is very low and it takes much time to engraft even if it is engrafted. Korean Patent Laid-Open Publication No. 2009-0113770 also discloses a method for producing heparin-bound fibrin gel, a kit for preparing a heparin-binding fibrin gel, a fibrin gel containing heparin-bound fibrinogen, There has been disclosed a possibility of applying the fibrin gel directly to the body to be used for the purpose of locally releasing a drug such as a growth factor protein or the like and being applicable to a tissue transplantation in a living body However, there is a disadvantage in that no study has been made in view of biomolecular tissue engineering.

Under these circumstances, the present inventors have intensively studied to develop a cell phyla that can be transplanted in vivo more effectively. As a result, fibrin gel having heparin-bound fibrinogen, thrombin, and cells were mixed to form a fibrin gel And the cells collected inside the fibrin gel are arranged so as to be oriented while the cells are cultured. Thus, a fibrin gel-type three-dimensional cell-free patch containing cells arranged in a directional direction can be easily transplanted in vivo And completed the present invention.

One object of the present invention is to provide a three-dimensional cellular edible patch.

Another object of the present invention is to provide a method for producing the three-dimensional cell-freezing patch.

It is still another object of the present invention to provide a wound treatment kit comprising the three-dimensional cell-freezing patch.

The present inventors intend to determine the conditions of the cell plant to develop a cell phyla more effectively in vivo. Generally, in terms of being transplanted into a living body, the cell plant should exhibit affinity for cell and organism, and exhibit binding affinity for a drug capable of promoting cell proliferation. The present inventors have searched for a supporter that meets the above conditions, and have focused on fibrin gels prepared using heparin-bound fibrinogen and thrombin. The fibrin gel is a supporter formed by the reaction of fibrinogen and thrombin, and has a very low density of solids. Therefore, the fibrin gel can capture cells inside the gel rather than the outer surface of the gel. In this case, And has a high affinity for cells and organisms, and is associated with heparin, exhibiting a high level of binding affinity for various drugs such as growth factor proteins and the like in comparison with conventional fibrin gels. In vivo, Since it is decomposed naturally, it is characterized in that it can be easily transplanted into a living body. Thus, a fibrin gel in which a cell growth factor of a human is bound and a desired cell is captured inside is prepared, and a fibrin gel-like three-dimensional cell culture patch Respectively. The prepared three-dimensional cell edible patch not only promotes the growth of the cell during culturing but also prevents the combined cell growth factor from being diluted in the medium and continuously stimulating the growth of the cells in the fibrin gel, It is possible to continuously stimulate the growth of the cell after the edible patch is transplanted in vivo.

In addition, the fibrin gels collected in the inside of the desired cells were respectively prepared as a circular paper round tubular and a net tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular tubular three- . At this time, the three-dimensional cell-freezing patch repeatedly lifting one end of the fibrin gel by means of a sterilizing forceps or the like at the time of replacing the medium, The cells can be arranged to have a certain directionality. Through such lifting, the cells contained in the three-dimensional cell edible patch of the present invention are induced to have the same type of cell structure as that of the in vivo tissue such as muscle cells, neurons, vascular cells and the like. The three-dimensional cell-freezing patch of the present invention, which is manufactured to have the same cell structure as that of the in vivo tissue, improves the bio-affinity when it is transplanted into the living body, thereby enabling to more effectively repair the damaged tissue.

Therefore, the three-dimensional cell-freezing patch of the present invention can be used as an active ingredient of a therapeutic kit which can be prepared in a site-specific form on a damaged part of a living body and is transplanted into the damaged part, There will be.

In order to achieve the above object, the present invention provides a fibrin gel-like three-dimensional cell-eating food, characterized in that a cell growth factor is bound, cells are collected inside, and the collected cells are arranged to have a directionality. Provide patches.

The term "cell growth factor" of the present invention means a substance capable of promoting or supporting the growth of a desired cell.

In the present invention, the cell growth factor is bound to the fibrin gel using heparin and bound to the fibrinogen by using the fibrinogen. The cell growth factor is preferably, but not limited to, (VEGF), transforming growth factor-β (TGF-β), platelet-derived growth factor (VEGF), and the like, and more preferably a vascular endothelial growth factor , Fibroblast growth factor (FGF), angiopoietin-1, etc. may be used alone or in combination.

The term "heparin" of the present invention is a type of acidic polysaccharide having a sulfate group and exhibits a blood coagulation inhibitory action, and a compound in which D-glucosamine and D-glucuronic acid have alternating chains it means. In the living body, capillaries such as liver and lung are present in many organs and blood, and the molecular weight is about 20,000.

In the present invention, the heparin has a molecular weight of 1,000 to 20,000 and can be interpreted to mean that it is activated in the form of NHS-heparin, and is used to provide sustained-release properties of the drug bound to the fibrin gel.

The term "fibrin gel" of the present invention means a gel formed by the reaction of fibrinogen and thrombin. The fibrin gel is a supporter formed by the reaction of fibrinogen and thrombin and has a very low density of solids. When the cells are collected in the gel, the collected cells can be redirected and proliferated, and affinity And is associated with heparin to exhibit binding affinity for various drugs such as growth factor protein and fibrin gels compared to conventional fibrin gels and is known to be decomposed naturally in the body as time elapses.

In the present invention, the fibrin gel is prepared in such a form that a cell growth factor binds and a desired cell is collected therein. When the fibrin gel is cultured, the cells captured therein can be grown and proliferated Rather, the captured cells can be rearranged to have directionality.

The term "cell" of the present invention means a cell which is collected in a fibrin gel supporting the three-dimensional cell culture patch, grown and propagated in the fibrin gel, and can be rearranged to have a directionality. The cell may preferably be a cell capable of replacing the damaged tissue at the wound site to which the three-dimensional cell-freezing patch provided by the present invention is to be implanted, more preferably a cell having the same or similar characteristics A stem cell capable of differentiating into a cell or a stem cell capable of being differentiated into the cell, and most preferably a stem cell capable of differentiating into a cell having a stem cell, a vascular cell, a smooth muscle cell, a cardiomyocyte, a bone cell, a chondrocyte, Inducible pluripotent stem cells, myeloid stem cells, and the like.

Particularly, the cells included in the three-dimensional cell-freezing patch of the present invention are configured to have a directionality such as nerve cells, muscle cells, blood vessel cells, etc. which are present in vivo. (For example, when the medium is replaced, etc.), lifting of the fibrin gel by using means such as sterilized forceps, and so on. The three-dimensional cell Edible patches are a novel technology that can not be found in any conventional cell plant.

There is also known a method for preparing a cell-free phylum of the present invention in which a cell is added to a fibrin gel, a hyaluronic acid gel, an alginic acid gel, a hydrogel or the like, and the cells are cultured to collect cells in the support . However, as for the relationship between the support and the cells contained in these conventional cell organisms, the cells are randomly collected within the support, and are only proliferated therefrom, and have a specific orientation and the cells are arranged in the support It is not. This is because there is no way to induce orientation in cells cultured in the support. However, in the present invention, by repeatedly performing lifting as described above, gravity causes directionality in the cultured cells, and consequently, the cells proliferated and bound in the fibrin gel exhibit a uniform direction as a whole. In addition, in order to exhibit such a directionality, the lifting should preferably be repeatedly performed in the same manner in terms of the position of contacting the sterile forceps, the lifting direction, the time, and the like.

Meanwhile, the three-dimensional cell-freezing patch provided by the present invention may be a form in which another kind of cultured cells are further combined on the outer surface of the patch. Specifically, since the three-dimensional embryogenic patch has a form in which cells are collected inside the fibrin gel constituting the three-dimensional embryonic patch, the outer surface of the patch is inoculated with the same or different cells and co-cultured, The cells can be in a combined form. Cells bound to the outer surface of the patch may be combined before repeating the lifting described above, may be combined after repetition of lifting, and may be arranged to be oriented or non-directional .

According to one embodiment of the present invention, heparin is activated to obtain NHS-heparin (Example 1-1), and the resulting NHS-heparin is reacted with fibrinogen to obtain fibrinogen conjugated with NHS-heparin Example 1-2), a fibrin gel in which NHS-heparin-bound fibrinogen and free fibrinogen were mixed, and a muscle cell, a growth factor and thrombin were added and reacted to produce a fibrin gel to which growth factors were bound and muscle cells were collected Example 1-3). In addition, fibrin gels in which the growth factors were bound and muscle cells were collected were prepared in various forms and cultured (FIG. 2).

In addition, the above-described lifting was repeatedly performed while culturing the above-prepared fibrin gel to prepare a three-dimensional cell edible patch showing the shape of the supporter (FIG. 3) and cultured without lifting The structures of the fibrin gels cultured under the lifting procedure with the fibrin gel (FIGS. 4A and 4B) and the orientation of the cells collected on the fibrin gel were compared (FIG. 5). As a result, it was confirmed that the cells collected in the fibrin gel without lifting process showed no specific direction, but the cells collected in the cultured fibrin gel showed directionality in a specific direction.

Accordingly, it was found that the lifting process is an essential process in manufacturing the three-dimensional cell-freezing patch of the present invention.

The prepared 3-D cell embryo patches can be manufactured to have similar morphology and similar structure to the site requiring transplantation, and the growth factor immobilized on the fibrin gel can be used not only when inoculating, culturing and propagating cells in vitro, It also promotes the proliferation of cells after transplantation in vivo, thereby accelerating wound healing.

As another embodiment for achieving the above object, the present invention provides a method for producing the three-dimensional cell edible patch.

Specifically, a method for producing a three-dimensional cell edible patch of the present invention comprises the steps of: (a) mixing a cell growth factor, fibrinogen, thrombin and cells to obtain a fibrin gel; And (b) lifting the fibrin gel while the fibrin gel is being cultured.

At this time, the fibrinogen binds to the cell growth factor via the heparin to which the activated heparin binds. At this time, the activated heparin may be in the form of NHS-heparin obtained by reacting carbodiimide and N-hydroxysuccinimide to heparin, though not limited thereto; The fibrinogen may comprise 1: 1 to 1: 5 (w / w) of activated heparin-bound fibrinogen and free fibrinogen; The drug for promoting cell proliferation is not particularly limited, but a cell growth factor may be used. Preferably, vascular endothelial growth factor (VEGF), transforming growth factor-β, TGF-β Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and angiopoietin-1 may be used alone or in combination; The cells inoculated into the fibrin gel include, but are not limited to, nerve cells, vascular cells, smooth muscle cells, cardiac muscle cells, skeletal muscle cells, bone cells, cartilage cells, fibroblasts, embryonic stem cells, induced pluripotent stem cells, And the like.

Among the above methods, lifting of the fibrin gel is an important process for providing directionality to the cells collected in the fibrin gel. Generally, since a wound usually occurs in a tissue composed of a directional cell such as a nerve cell, a muscle cell, and a vascular cell, when a three-dimensional cell-free patch prepared to include cells exhibiting directionality is used, Can be restored. The time for performing the lifting process is not particularly limited. Preferably, the time for performing the lifting process can be performed while the medium is being replaced. The means for performing the lifting process is not particularly limited, but preferably, Sterile forceps can be used.

In addition, the method may further include a step of inoculating cells on the outer surface of the fibrin gel before culturing the fibrin gel in the step (b). When the fibrin gel is cultured after cells are inoculated on the outer surface of the fibrin gel, the cells collected inside the fibrin gel and the cells inoculated on the outer surface can be independently cultured. Therefore, a single fibrin gel A three-dimensional cell-based patch containing two kinds of cells can be produced. At this time, the cells inoculated on the outer surface of the fibrin gel may be the same as or different from the cells collected inside the fibrin gel; The fibrin gel may be inoculated before the fibrin gel is cultured, or may be inoculated after the fibrin gel is cultured and then cultured again. Wherein the cells inoculated on the outer surface are arranged to be directional by lifting when the fibrin gel is inoculated before the fibrin gel is cultured and if the cells inoculated on the outer surface are inoculated after the fibrin gel is inoculated, And may be arranged so as not to have directionality.

Meanwhile, the method for producing a three-dimensional cell-freezing patch of the present invention is manufactured in a standardized form, and can be applied to various wounds for general application, and can be tailored to wounds, Can be applied. For example, a three-dimensional cellular edema patch that is designed to capture muscle cells or fibroblasts and have a fixed-sized cube shape can be used to treat most skin or muscle damaged wounds. In addition, in order to treat wounds in which a part of the skin and dermal layer is lost by wound or the like, a three-dimensional cell culture patch in which muscle cells or fibrous cells are collected in a form similar to the lost region is prepared, By transplanting, the wound can be treated.

As another embodiment to achieve the above object, the present invention provides a wound treatment kit comprising the three-dimensional cell-freezing patch as an active ingredient.

The term "wound treatment" of the present invention refers to any act of protecting the damaged cell, such as a drug treatment for restoring damaged skin cells, muscle cells, nerve cells, vascular cells, etc. by physical factors such as wound, .

As described above, the three-dimensional cell edible patch of the present invention can be produced so as to have the same shape and the same structure as the intended wound site, and thus can be used as an effective ingredient of a kit for treating a wound.

Thus, the wound treatment kits of the present invention may further comprise suitable carriers, excipients or diluents conventionally used to maintain the shelf life and cell viability of the three-dimensional cell edible patches. In addition, the kit may be prepared in the form of injections, implants, etc. The three-dimensional cell-freezing patch, which is an effective ingredient of the wound treatment kit of the present invention, Therefore, it is preferable to implant in a form to be directly introduced into a wound site.

According to another aspect of the present invention, the present invention provides a method for treating a wound including implanting a wound treatment kit including the three-dimensional cell-freezing patch, into a scarred area of an individual do.

The term "individual" in the context of the present invention includes, without limitation, mammals including rats, cattle, humans,

In the method of treating a wound of the present invention, the implantation of the kit can be performed through any conventional pathway as long as it can reach the target tissue. The wound treatment kit of the present invention is not particularly limited, but may be implanted into an intravascular, intramuscular, subcutaneous, or intradermal route according to the purpose.

The three-dimensional cell-freezing patch provided by the present invention can be manufactured to have the same shape and the same tissue as the injured wound area, so that it can be utilized for more effective wound treatment.

1 is a graph showing changes in the level of release of VEGF or angiopoietin-1 according to the type of fibrin gel.
Fig. 2 is a photograph showing the shape of a circular fibrin gel in the form of a round paper and a tubular fibrin gel in the form of a net tube.
FIG. 3 is a schematic view showing a method of imparting a directionality when a three-dimensional cell-freezing patch is manufactured using a tubular fibrin gel.
FIG. 4A is a photograph showing the result of immunostaining of a cell-type plant in which a muscle cell is cultured in a round fibrin gel.
FIG. 4B is a photograph showing the result of immunostaining of a myeloma cell line in which a muscle cell is cultured in tubular fibrin gel.
FIG. 5 is a graph showing a directional change of a three-dimensional cell-freezing patch according to whether or not the lifting process is carried out. In FIG. 5, black squares represent the direction of cells collected on a round fibrin gel, and red squares are collected on a tubular fibrin gel Indicating the orientation of the cell.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Preparation of fibrin gel containing growth factor

Example  1-1: Preparation of activated heparin

100 mg (0.00002 mol) of low molecular weight heparin (4000-6000, Sigma) was added to 1 ml of MES buffer ([2- (N-morpholino) ethane sulfonic acid] buffer, 0.05 M, pH 6.0) After completely dissolved, 0.0046 g of NHS (N-hydoroxysuccinimide, 0.00008 M) and 0.015336 g of EDC (N- dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride, 0.004 M) To prepare an NHS-heparin solution. The prepared NHS-heparin solution was precipitated with anhydrous acetone, extracted and lyophilized for 24 hours to prepare activated heparin.

Example  1-2: Activated heparin Combined  Obtaining fibrinogen

The activated heparin (60 mg) and human plasma-derived fibrinogen (100 mg) prepared in Example 1-1 were dissolved in 20 ml of PBS (phosphate buffered saline, pH 7.4) and reacted at 4 ° C for 3 hours. At this time, when fibrinogen was dissolved, it was slowly dissolved so as not to cause foaming. After the completion of the reaction, anhydrous acetone was added to the reaction solution for precipitation and extraction, and lyophilized for 24 hours under light-shielding conditions to obtain heparin-bound fibrinogen. The heparin-bound fibrinogen thus obtained was dissolved in 20 ml of phosphate buffer and dialyzed with dialysis membrane (MWCO: 12-14,000) at 4 ° C for 24 hours while exchanging distilled water five times or more. Thus, fibrinogen Was eluted and again lyophilized under light-shielding conditions for 48 hours to obtain heparin-bound fibrinogen in the form of a white powder.

Example  1-3: Preparation of fibrin gel

First, human vascular endothelial growth factor (VEGF) or angiopoietin-1 was dissolved in an aprotinin solution used as a protease inhibitor.

Next, the mixture of heparin-bound fibrinogen obtained in Example 1-2 and fibrinogen separated from human plasma was mixed at a ratio of 1: 2 (w / w) to obtain a mixture.

In addition, muscle cells to be collected in fibrin gel were prepared by culturing.

Finally, human plasma-derived thrombin (10 mg) was added to and dissolved in a solution (1000 μl) in which calcium chloride was dissolved to obtain a thrombin solution.

The vascular endothelial growth factor (VEGF) or angiopoietin-1 is added to the obtained aprotinin solution by adding the fibrinogen mixture and the cultured muscle cells, and then adding the thrombin solution to cross- And fibrin gel (HCF) in the form of muscle cells were prepared.

On the other hand, as a control, fibrin gel prepared by adding fibrinogen and thrombin separated from human plasma to the obtained aprotinin solution, and fibrinogen, heparin and thrombin separated from human plasma in the obtained aprotinin solution (Fibrin gel + free heparin) were prepared.

The prepared three kinds of fibrin gels (HCF, fibrin gel and Fibrin gel + free heparin) were immersed in 1 ml of phosphate buffer and the levels of VEGF or angiopoietin-1 released from each of the fibrin gels were measured by ELISA For 30 days (Fig. 1). 1, it was confirmed that fibrin gel (HCF) prepared by the method of the present invention can release VEGF or angiopoietin-1 in a sustained manner, unlike other fibrin gels used as a control group.

Example  2: Fabrication of 3-D Cellular Patch Using Fibrin Gel

The muscle-derived cells were inoculated into the fibrin gel containing the growth factor prepared in Example 1 and cultured to prepare edible patches of the three-dimensional muscle cells.

First, round type fibrin gels in the form of round paper and tubular fibrin gels in the form of net tubes were prepared by applying the method of Example 1 (FIG. 2).

Next, the muscle cells derived from the muscle were inoculated into each of the fibrin gels contained in the culture broth together with the culture solution, and cultured for 7 days while changing the medium every two days. At this time, the tubular type fibrin gel was lifted by using the sterilized forceps when the medium was replaced (FIG. 3), and the round type fibrin gel did not perform the lifting.

After completion of the incubation, each of the fibrin gels was immunostained and the fibrin gel was divided into upper, middle, and lower layers in the Z direction, and each layer was photographed using a fluorescence microscope (FIGS. 4A and 4B ).

As shown in FIGS. 4A and 4B, when the tubular fibrin gel was used to make a three-dimensional cell culture patch, a tubular-type muscle fiber bundle having a uniform orientation similar to that of muscle tissue and having a uniform size (thickness of 50 μm) biomimetic fiber bunch). Since the produced muscle fiber bundle has a thickness of about 50 탆 or less, supply of oxygen and nutrients from outside can be performed well to improve cell growth and reduce cell death The results of this study are as follows. On the other hand, it was confirmed that the cells collected on the round fibrin gel showed no directionality.

In addition, the captured images were applied to a computer image analysis program, image J (NIH, US), to confirm the directions of the muscle cells present in the respective layers (FIG. 5). The orientation of the muscle cells may be determined in any direction of the round fibrin gel or in the range of -90 degrees to +90 degrees with respect to the angle of the cells tilted to the right (+) or left (-) with respect to the longitudinal direction of the tubular fibrin gel The number of cells corresponding to the measured angle was counted and displayed, and the angle of the cell was measured as the angle formed by the long axis of the cell with respect to the baseline.

As shown in FIG. 5, the cells (black squares) collected in the round fibrin gel are distributed uniformly with the direction of the entire region (range of -90 to +90 degrees), while the cells collected in the tubular fibrin gel (Red squares) show that 80% or more is concentrated in a certain region (range of -20 degrees to +20 degrees).

Therefore, the lifting of the fibrin gel was found to be an essential step in the preparation of the three-dimensional cell culture patch of the present invention including cells arranged to have directionality by adding directionality to the cells.

Claims (16)

Wherein the cell growth factor is bound, the cells are collected inside, and the collected cells are arranged so as to be directional.
The method according to claim 1,
Wherein the cell growth factor binds with fibrinogen constituting the fibrin gel through heparin.
3. The method of claim 2,
Wherein the heparin has a molecular weight of 1,000 to 20,000 and is activated in the form of NHS-heparin.
The method according to claim 1,
The cell growth factor may be selected from the group consisting of vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-), platelet-derived growth factor (PDGF) Wherein the growth factor is a growth factor selected from the group consisting of fibroblast growth factor (FGF), angiopoietin-1, and combinations thereof.
The method according to claim 1,
Wherein the cell growth factor is released from the fibrin gel in a sustained release form.
The method according to claim 1,
Wherein the cell is selected from the group consisting of a neuron, a vascular cell, a smooth muscle cell, a cardiac muscle cell, a bone cell, a chondrocyte, a fibroblast, an embryonic stem cell, an induced pluripotent stem cell, a bone marrow stem cell, / RTI > is a three-dimensional cellular edible patch.
The method according to claim 1,
Wherein the cells are further bound to the outer surface of the patch.
(a) mixing a cell growth factor, fibrinogen, thrombin and cells to obtain a fibrin gel; And
(b) repeatedly performing lifting of the fibrin gel while culturing the obtained fibrin gel.
9. The method of claim 8,
The cell growth factor may be selected from the group consisting of vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-), platelet-derived growth factor (PDGF) A fibroblast growth factor (FGF), and combinations thereof.
9. The method of claim 8,
Wherein the fibrinogen is a mixture of fibrinogen and free fibrinogen to which the activated heparin is bound.
11. The method of claim 10,
Wherein the ratio of activated heparin-bound fibrinogen to free fibrinogen contained in the fibrinogen mixture is 1: 1 to 1: 5 (w / w).
11. The method of claim 10,
Wherein the activated heparin-bound fibrinogen is capable of binding NHS-heparin to bind to a cell growth factor.
9. The method of claim 8,
Wherein the cell is selected from the group consisting of a neuron, a vascular cell, a smooth muscle cell, a cardiac muscle cell, a bone cell, a chondrocyte, a fibroblast, an embryonic stem cell, an induced pluripotent stem cell, a bone marrow stem cell, .
9. The method of claim 8,
Further comprising inoculating cells to the outer surface of the fibrin gel prior to culturing the fibrin gel in step (b).
9. The method of claim 8,
Further comprising the step of culturing the fibrin gel in the step (b), inoculating the cells on the outer surface of the fibrin gel, and then re-culturing the cells.
A wound treatment kit comprising the three-dimensional cell-freezing patch of any one of claims 1 to 7.

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