KR20130127329A - Method for preparing concentrated cell culture medium via 3d-culture and use thereof - Google Patents

Abstract

The present invention relates to a method for preparing a culture medium containing growth factors or cytokines, comprising steps of attaching adult stem cells on a scaffold and performing three-dimensional culture; a concentrated culture liquid prepared by the method; a composition containing the concentrated culture liquid as an active ingredient for generating bone tissues; a method for preparing a transplant for generating bone tissues, which contains a culture liquid-loaded scaffold for transplantation; and the transplant for generating bone tissues, containing the culture liquid-loaded scaffold for transplantation. The method for preparing the transplant comprises the steps of: preparing the culture liquid; and loading the culture liquid on the scaffold for transplantation. [Reference numerals] (AA) Concentrated culture liquid;(BB) Cell

Description

Method for preparing highly concentrated culture medium through three-dimensional culture of stem cells and use of highly concentrated culture medium {Method for preparing concentrated cell culture medium via 3D-culture and use}

The present invention comprises a method for producing a culture medium containing a growth factor or cytokine, comprising the step of attaching the adult stem cells to the scaffold, the concentrated culture medium prepared by the method, the concentrated culture medium as an active ingredient It relates to a bone tissue regeneration composition comprising, a method for producing a bone tissue regeneration implant comprising the step of loading the composition into a graft scaffold and to a bone tissue regeneration implant comprising a graft scaffold prepared by the method .

Bone tissue can be damaged by various diseases, including spinal cord injury, fractures, infections, and tumors. In the case of a simple fracture, natural treatment is possible, but if a bone loss occurs beyond the natural treatment limits, a treatment for treating and / or regenerating it is necessary. In addition, the extraction of alveolar bone occurs due to periodontal disease or trauma, which causes problems in prosthetic treatment and implant treatment. Particularly, successful implants require a fixed length and diameter medium to be implanted, but implantation may be restricted in the case of lack of alveolar bone. These sites require bone grafts to increase the height and width of the alveolar bone, and several regenerative therapies have been developed for this purpose. The most widely used for such bone defects are bone grafts and autologous bone, allogeneic bone, xenograft or synthetic bone is used as a graft material for bone regeneration. Autologous bone contains bone cells capable of direct bone formation and is the best bone graft without rejection, which guarantees a high success rate, but requires additional surgery on the harvesting site for bone extraction and concomitant postoperative discomfort. It is not easy to collect as much as necessary because of the limited amount. Therefore, in clinical practice, there is an increasing demand for the development of bone graft material to replace autologous bone. Allogeneic bone has been widely used as a substitute for autologous bone because it has been reported to be capable of extracting a large amount of bone compared to autologous bone graft and has the advantage of being able to be transplanted with a single operation and has a high possibility of bone formation. However, it is not easy to manipulate and does not show consistent bone regeneration results, and may cause failure in bone regeneration, or may cause side effects such as immune rejection reactions, and the possibility of spreading disease due to infection when bones collected from a disease holder are used. This has been controversial. Heterogol is a bone bone used to treat bone tissue collected from animals other than humans and used as an implant. The deproteinization process to remove the antigenicity and infection potential of xenografts is carried out, and the right bone that has undergone this deproteinization process is widely used for bone transplantation in the dental field. In addition, various synthetic materials that can be used as synthetic bones are being developed. To be used as synthetic bone, it must be non-toxic, biocompatible, porous, and regenerated bone cells that can penetrate stably, proliferate, and decompose in the body over time.

On the other hand, as the stem cell isolation and culture technology is developed, a method of transplanting stem cells capable of differentiating into various tissues into the affected area as a cell therapy agent is also used. Cell therapy products should also satisfy conditions similar to the bone graft material. After transplantation, the cells must exhibit the intrinsic function of the desired cells, be safe and free of immune rejection, and a large amount of cells should be secured for transplantation therapy. Therefore, autologous transplantation is premised or in case of allograft, it is used as a tissue conformation form. As such, cell therapy agents may be classified into autologous, allogeneic, and heterologous cell therapy, depending on the origin of the cells. Autologous cell therapy isolates cells from the patient and simply proliferates them in an undifferentiated state in vitro, or induces differentiation and development while incubating, or changes the traits of the cells by injecting certain functions to give them specific functions. Transplant. On the other hand, allogeneic cell therapy is a cell therapy that undergoes a similar process to autologous cells but separates cells from other healthy donors, simply multiplies them in vitro, and transplants them into patients. Using cells.

The most easily performed is allogenic grafts using allogeneic cell therapy. However, the biggest disadvantage of allografts is the immune rejection reactions caused by transplantation of cells from other individuals. In some cases, autologous transplantation is possible (bone marrow, epithelial, and hematopoietic stem cells), but if this is impossible or difficult, it is inevitable to perform allografts, but this is the factor that most restricts the application of stem cells. It is a challenge that must be addressed to expand its application. Nevertheless, allogeneic transplantation of adult stem cells has been used as an alternative to overcome the problem of autologous transplantation. It can relieve the burden on the patient, such as the limitation of the time required for in vitro culture after collection, which is necessary for autotransplantation, the economic constraints caused by the high value-added cost and the surgical procedure that must be preceded. Research and development is underway to eliminate the risk of immune rejection, although it is insignificant. Currently, it is based on differentiation through in vitro culture and infused into a differentiated state on the premise of a tissue-compatible transplant. . However, transplantation into such differentiated cell types blocks the possibility of differentiation into various cells, which is a fundamental advantage of stem cells, and at the same time significantly reduces the secretion of cytokines that can induce cell activation in the surrounding host system. Fusion and activation ability are reduced.

The present inventors have combined the advantages of transplanting the bone graft material and stem cell therapy to find a way to regenerate bone tissue efficiently without worrying about the immune rejection reaction, and as a result, the stem cells are attached to the 3D porous scaffold and cultured. By implanting a biodegradable scaffold loaded with a concentration of a growth factor and a cytokine-containing culture solution obtained by the transplantation, it was confirmed that the stem cells were directly loaded and had a higher bone regeneration effect than when transplanted.

One object of the present invention is to provide a method for producing a culture medium comprising a growth factor or cytokine comprising the step of attaching the adult stem cells to the scaffold three-dimensional culture.

Another object of the present invention is to provide a concentrated culture prepared by a method for producing a culture medium containing a growth factor or cytokine comprising the step of attaching the adult stem cells to the scaffold three-dimensional culture.

Another object of the present invention to provide a composition for bone tissue regeneration comprising the concentrated culture as an active ingredient.

Still another object of the present invention is to prepare a culture medium by a method for producing a culture medium comprising a growth factor or cytokine comprising the step of attaching the adult stem cells to the scaffold three-dimensional culture; And it provides a method for producing a bone tissue regeneration implant comprising a scaffold for loading the culture medium, comprising the step of loading the prepared culture solution in the scaffold for transplantation.

Still another object of the present invention is to provide an implant for bone tissue regeneration comprising a scaffold for transplantation loaded with the prepared culture solution.

As one aspect for achieving the above object, the present invention provides a method for producing a culture medium comprising a growth factor or cytokine comprising the step of attaching the adult stem cells to the scaffold three-dimensional culture.

The term "adult stem cells" of the present invention is a cell capable of self-proliferation, which is a characteristic of general stem cells, and having the ability to differentiate into a variety of cells through suitable environment and stimulation, that is, 'undifferentiated cells'. Cells with the potential to develop into specialized cells and can be isolated from bone marrow, skeletal muscle, adipose tissue, umbilical cord blood, umbilical cord, skin, amniotic membrane, placenta and the like. In particular, mesenchymal stem cells are the cells that assist in the formation of tissues such as cartilage, bone, fat, bone marrow, epilepsy, muscle, and nerves. In adults, they remain in the bone marrow, but are also present in cord blood, peripheral blood, and other tissues It means the cell which can be obtained from these. Preferably, the adult stem cells may be bone marrow-derived mesenchymal stem cells, but is not limited thereto. The method for obtaining the adult stem cells may be by a method known in the art, and is not limited to the method of the embodiment of the present invention.

The term "scaffold" of the present invention refers to a solid support having a constant skeleton, and includes without limitation as long as stem cells can be attached to the scaffold and proliferated and cultured. Preferably the scaffold may be a collagen scaffold, but is not limited thereto. Such collagen scaffolds can be used as a porous solid support.

In the general cell culture method, three-dimensional culture means a method of culturing in a suspended state in a liquid medium without being attached to a culture dish, but the term "three-dimensional (3D) culture" of the present invention is attached to a three-dimensional porous solid support culture. As the cells proliferate, it refers to a method of culturing in three dimensions while filling the pores in the support. In a specific embodiment of the present invention, the collagen scaffold is used as a solid support, but is not limited thereto. Through the three-dimensional culture, the method of the present invention uses a small amount of culture medium that cannot be obtained in the conventional two-dimensional culture of stem cells, so that a high concentration of the growth factor or cytokine can be obtained at a low concentration time. have.

The term "growth factor" of the present invention means a naturally occurring substance capable of promoting cell growth, proliferation and differentiation. Generally a protein or steroid hormone, it plays an important role in the regulation of various cellular processes. It acts as a signaling molecule between cells, and examples thereof include cytokines and hormones that bind to specific receptors on the surface of target cells. Sometimes differentiation and development are promoted depending on the growth factor. For example, bone morphogenic proteins promote bone cell differentiation, while fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) are known as vascular differentiation (angiogenesis). promote angiogenesis Although often mixed with cytokines, cytokines are involved in hematopoietic and immune cells. Growth factors have a positive effect on cell division, while cytokines are a neutral term for the effect of molecules on proliferation. Preferably, the growth factor is IGF-1 (insulin-like growth factor-1), TGF-β1 (transforming growth factor-beta 1), bFGF (basic fibroblast growth factor), BMP2 (bone morphogenetic protein 2) or VEGF (vascular) endothelial growth factor), but is not limited thereto.

The term "cytokine" of the present invention is a small cell-signaling protein molecule secreted by many cells and refers to a signaling molecule widely used for intercellular communication. It can be classified as a protein, peptide or glycoprotein, and the term cytokine includes both large and diverse family of regulators produced throughout the body by cells of various developmental origins. However, in general, the term is used to refer to immunomodulating agents such as interleukin and interferon. Preferably, the cytokine may be CXCL2 (chemokine C-X-C motif ligand 2) or IL-8 (interleukin-8), but is not limited thereto.

The term "culture medium" of the present invention refers to the remaining solution after removing the cells after culturing the stem cells in a liquid medium to support the growth and survival in vitro of the isolated stem cells, the cells during the culture period Proteins, such as growth factors and cytokines secreted from, and all the nutrients consumed when culturing the cell are included. The medium includes all the usual medium used in the art suitable for the culture of adult stem cells, the medium and culture conditions can be selected according to the type of cells. The medium used for the culturing is preferably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source and a trace element component. Such cell culture minimal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI1640, F-10, F-12, α Minimal Essential Medium (αMEM), Glass Minimal Essential Medium (GMEM) and Iscove's Modified Dulbecco's Medium (IMDM), but are not limited thereto. In addition, the medium may be added an antibiotic such as penicillin, streptomycin or gentamicin, or serum additives such as fetal bovine serum (FBS) or human serum albumin. It can be included as.

The present invention may further comprise the step of collecting and concentrating the culture. The step of collecting the culture may be performed by a method known in the art, for example, may be collected by centrifugation. Most of the cultures collected as described above are bulky as liquid components and thus are not easily stored. Thus, further concentration may be performed. The concentrated culture solution may be aliquoted and stored frozen, thawed as necessary, and diluted to a desired concentration. Concentration of the protein can be carried out by methods known in the art, such as using a protein concentration filter. In a specific embodiment of the present invention, using a protein concentration filter, while maintaining the protein content, by reducing the volume of 1.5 ml culture medium to 30 μl it was possible to obtain a concentrated culture medium significantly increased the concentration of growth factors and cytokines.

The present invention may further comprise the step of culturing by giving electric stimulation. The process is to promote the secretion of growth factors and cytokines can be carried out by methods known in the art of electrical stimulation, it is preferable to install the electrode on the cell culture plate to adhere the cells on the electrode and culture Stimulation can be applied to the flow, but is not limited thereto.

The present invention may further include the step of collecting and concentrating the culture solution of the cultured cells by giving additional electrical stimulation as mentioned above.

In a specific embodiment of the present invention, it was confirmed that gene expression and secretion of bone growth factors related to growth factors related to bone regeneration and cytokines related to stem cell migration were increased only by attaching to the collagen scaffold (Example) 2 to 4, FIGS. 4 and 5). Furthermore, by concentrating using a protein concentration filter, the protein can be concentrated to a concentration of about 50 times higher with little loss of protein (Table 2) (Example 5). The concentrated culture solution thus prepared is capable of inducing differentiation into excellent bone cells. It was confirmed that the (Fig. 8 and 9).

As another aspect, the present invention provides a concentrated culture prepared by a method for producing a culture medium comprising a growth factor or cytokine comprising the step of attaching the adult stem cells to the scaffold and three-dimensional culture.

As another aspect, the present invention provides a composition for bone tissue regeneration comprising the concentrated culture as an active ingredient.

The term "osseous tissue" of the present invention is a term referring to the tissue forming the bone. Bones are made of dense bone on the surface, and both ends of the center and long bones are composed of spongy bone associated with mesh. Most bones initially develop as cartilage in connective tissue and later into bone tissue, some of which are produced directly from connective tissue. At the distal end, there is a joint surface in contact with neighboring bones, the surface of which is covered with articular cartilage, a supernatural bone. The sponge shochu in the sponge is characterized by a certain arrangement. Osteoblasts are arranged between the bone lamina and are connected to other bone cells adjacent to each other with a protuberant projection to an irregular star. On the surface of the bone is a tough connective tissue periosteum, and nerves and blood vessels are distributed to protect and nourish the bone. Defects of the periosteum make it difficult to survive, develop, and regenerate bones. The bone component is 20% moisture, 35% organic matter including cells, 45% minerals, because the bones maintain a certain elasticity because there is an organic material. As age increases, minerals (mainly calcium phosphate) increase and bone hardness also increases.

The term "regeneration" of the present invention generally refers to an action in which an organism rebuilds or restores its function when a part or function of a body is lost. This regenerative capacity is stronger when the system is simpler and the degree of evolution is lower systematically. The present invention in a specific embodiment using a culture medium mixed 1 or 5% of the concentrated culture medium of the present invention in a culture medium for inducing differentiation of osteoblast differentiation, higher ALP activity (FIG. 8) and calcification than a medium for osteoblast differentiation ( 9) confirming that the culture solution obtained from the three-dimensional culture of the present invention to efficiently reproduce bone tissue, it was confirmed that it can be used as a composition for bone tissue regeneration.

As another aspect, the present invention comprises the steps of preparing a culture medium by a method for producing a culture medium comprising a growth factor or cytokine comprising the step of attaching the adult stem cells to the scaffold three-dimensional culture; And it provides a method for producing a bone tissue regeneration implant comprising a scaffold loaded with the culture, comprising the step of loading the prepared culture solution into the scaffold for transplantation.

The term "transplantation" of the present invention generally refers to the process of transferring the donor's cells, tissues or organs to the damaged tissue or organ of the recipient. Depending on the donor type, autografts transplanted their own cells or tissues elsewhere, allografts (eg, from humans to humans) that transplant cells, tissues, organs from a donor that is the same species as the recipient Can be classified as xenografts that transplant organs from a species (e.g., organs of an animal to humans). The subject of transplantation includes, without limitation, any cell, tissue or organ of the donor, as well as any material that can aid the regeneration of the injured tissue of the recipient. The transplantation can be performed by a method known in the art, and is not limited to the embodiment of the present invention. For example, surgery can be performed with surgery, and materials such as cells can be injected directly into the affected area.

The term "graft scaffold" of the present invention is a structure that serves to support the material to be implanted to stay in the structure to be implanted with the material to be implanted. Therefore, it should be a substance having biocompatibility and high cell or tissue engraftment rate. Preferably the transplant scaffold may be a biodegradable material, more preferably collagen.

The term "loading " of the present invention refers to the process of containing a therapeutically effective substance in the implantable scaffold. Any method that satisfies such a function can be used without limitation, and in the present invention, the stem cell concentrate culture medium effective for bone tissue regeneration is applied to and absorbed into the collagen scaffold used as a transplant scaffold.

The term "graft" of the present invention is a support for isolating a damaged site from the outside or containing a transplanted cell or secreted therapeutic material and allowing the cell or therapeutic material to remain, and refers to a substance that can be implanted in a human body or a mammal. it means. Such implants include, without limitation, various materials used in the art, such as synthetic polymers and natural materials having biodegradability as a support for tissue engineering. Preferably may be collagen. Since the implant of the present invention is a collagen scaffold containing a stem cell concentrated culture medium and does not contain non-magnetic cells, it was confirmed in a specific example that there is no immune rejection reaction and no inflammatory reaction (FIG. 11). Therefore, even without the administration of a separate immunorejection inhibitor or anti-inflammatory agent to suppress the immune rejection or inflammatory response that may occur when various implants are implanted in the body, the transplanted transplant is stable without immune rejection or inflammation in vivo It can engulf as. The implant of the present invention is a collagen scaffold comprising a stem cell concentrated culture medium, and may further include mesenchymal stem cells in addition to the collagen scaffold including the concentrated culture medium.

As another embodiment, the present invention provides Provided is an implant for bone tissue regeneration comprising a scaffold loaded with the prepared culture solution.

A transplant consisting of a collagen scaffold containing a growth medium obtained by attaching the stem cells of the present invention to a solid support and a culture medium containing a large amount of cytokines and a concentrated culture solution, is transplanted into a rat cranial tube defect animal model. (FIG. 10) promoted the migration of stem cells (FIG. 11) without an inflammatory response (FIG. 11) and showed higher bone regeneration ability than the control group transplanted with stem cells (FIGS. 13-15).

The culture solution obtained by attaching the stem cells of the present invention to the solid scaffold and carrying out three-dimensional culture contains a large amount of growth factors and cytokines, and the concentrated culture solution is loaded onto the collagen scaffold and transplanted to the bone defect site to obtain a high inflammatory response. It was confirmed that the bone regeneration effect. Therefore, the concentrated culture solution of the present invention does not contain cells and thus may be utilized as a composition for bone regeneration without worrying about immunorejection or inflammatory reactions.

Figure 1 shows the actual appearance (right) of the electrical stimulation device (in the middle) of the in vitro culture system for electrical stimulation and the electric stimulation intensity control device (left) in the abnormal current stimulator and the collagen cultured cells in the electrical stimulation device (right) It is also.
2 is a diagram showing an electron micrograph for confirming cell adhesion to the scaffold. The collagen scaffolds to which cells are not attached, the scaffold to which cells are attached, and the experimental group (EC) to which cells are attached and electric stimulation are applied to the scaffolds from the left, respectively.
Figure 3 is a diagram showing the gene expression of growth factors and cytokines through real-time RT-PCR (real-time RT-PCR). The expression level of each gene was compared and shown in 2D culture and 3D culture.
Figure 4 is a diagram showing the number of culture days by comparing the amount of gene expression according to the presence or absence of electrical stimulation in the three-dimensional culture.
5 is a diagram showing the results of enzyme linked immunosorbent assay (ELISA) for comparing the expression levels of growth factors and cytokines according to the culture method. The expression levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin-8 (IL-8) were measured, and the expression levels according to 2D and 3D cultures and the presence or absence of electric stimulation in each of them. Was measured and compared.
Figure 6 is a diagram showing a protein concentration filter used for the concentration of the culture.
Figure 7 is a diagram showing the results of MTT analysis for confirming the cytotoxicity of the concentrated culture solution. Cell viability was measured for the cells cultured in the normal culture solution in which the concentrated culture solution of the present invention was mixed at 1, 2.5 and 5% for 3 days.
8 is a diagram showing ALP (alkaline phosphatase) activity according to the culture conditions. Concentrated cultures prepared by 3D culture in the presence or absence of electric stimulation in the control (con; ascorbic acid, β-glycerophosphate), conditions to differentiate into bone cells (dex; dexamethasone) and conditions to differentiate into bone cells ALP activity was measured for experimental groups cultured with the indicated concentrations to determine the effect on osteoblast differentiation.
Figure 9 is a diagram showing the result of quantifying the alizarin red S (alizarin red S) staining results and the amount of the alizarin red S obtained by decolorizing to confirm the effect of the concentrated culture on osteoblast differentiation. Stem cells cultured in each condition stained with Alizarin Red S were photographed and then decolorized with a decolorizing solution, and the absorbance of the solution was measured at a wavelength of 595 nm and converted into concentration.
10 is a view showing the preparation of cranial canal damage experimental animal model and the animal experiment using the same.
Figure 11 is a view showing the H & E staining results for confirming the inflammatory response by the concentrated culture.
12 is a diagram showing the results of stem cell migration promotion experiment of the concentrated culture solution. The collagen scaffold loaded with the normal culture solution and the concentrated culture solution of the present invention was inserted into the cranial canal damage experimental animal model, and injected with human bone marrow-derived stem cells labeled with the fluorescent dye Dil. As a comparative experiment group, a scaffold loaded with stromal cell-derived factor-1 (SDF-1), which is a stem cell migration promoter, was used.
13 is a view showing the results of quantitative analysis of the degree of bone formation by micro CT imaging. The control cell transplanted with stem cells loaded on the scaffold, the concentrated culture medium prepared with 3D culture, the experimental group loaded with scaffold loaded with media, and the concentrated culture medium prepared by 3D culture with electric stimulation were applied. Bone volume (BV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) for the media-EC implanted scaffold And trabecular thickness (Tb.Th).
14 is a view showing an image of the new bone tissue measured using a micro CT. Each implant was implanted into a cranial canal damaged animal model and 4 weeks later, micro CT was used to image newly formed bone tissue.
15 is a diagram showing the histological staining results for checking the degree of bone formation. Each implant was implanted into a bone injury animal model that caused a 5 mm diameter bone defect in the cranial canal and histologically evaluated the degree of bone formation using Masson's tricolor staining.

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

Example  1: human Bone marrow origin Intermediate lobe Adult stem cell  Isolation and Culture

Human bone marrow as an adult stem cell source of the present invention was obtained with an inhalation puncture instrument in the iliac bone with the consent of the patient, and obtained permission from the institutional review board (IRB) of the university. Bone marrow fluid was extracted from the iliac crest site of bone marrow donors by the method described in Vilamitjana-Amedee et al. (In Vitro Cell Dev. Biol. Anim., 1993, 29A (9): 699-707). Ficoll solution (Ficoll-Paque ^TM PLUS) was added to separate the layers according to the density to separate the nucleated cell layer formed between the supernatant and the lower blood layer. The isolated nucleated cells were cultured in medium conditions containing 10% fetal bovine serum (FBS) in DMEM (Dulbecco's modified Eagle's medium) at a density of 3 to 4 × 10 ⁴ cells / cm ² . Once every 4 days with fresh medium, a colony of cells was formed and propagated to 60-70% of the culture dish, followed by subculture. In order to secure a sufficient number of cells, the number of passages was increased and the experiment was performed in the second passage and the remaining cells were cryopreserved.

Example  2: electrical stimulation and planar or steric cell culture conditions

As a method for promoting the secretion of growth factors and cytokines in isolated stem cell cultures, the cells were cultured in three dimensions by attaching them to the culture and / or steric scaffold while giving electric stimulation. The device designed to give electric stimulation during the culture is shown in FIG. 1. The electrode was used as a plate deposited with gold, and the cells were adhered to the channel electrode. Electrical stimulation was applied in combination with stereoculture conditions. 2D planar culture was cultured in a general Teflon culture dish, whereas for 3D culture, a collagen scaffold was introduced and attached to the culture. The electrical stimulation was given differently according to the culture conditions. In the 2D planar culture, seeding was performed at a cell density of 1.7 × 10 ³ cells / cm ² , and then stimulated for 4 days under conditions of 1.5 μA, 250 μs, and 100 Hz from the next day. The cells were attached to the collagen scaffold, and in 3D culture, 5 × 10 ⁵ cells were seeded in 5 mm collagen at a volume of 20 μl, allowed to attach for 3 to 4 hours, and then the culture solution was added and The cultures were stimulated for 4 days at 20 μA, 250 μs, and 100 Hz. On the 4th day of cultivation, the surface of the collagen scaffold used for 3D culture and the experimental group to which human bone marrow-derived adult stem cells were attached, and the experimental group to which adult stem cells were attached to the collagen scaffold and subjected to electrical stimulation were injected on the 4th day of culture. It was confirmed by scanning electron microscopy.

As a result of scanning electron microscopy, it was confirmed that the surface of the collagen scaffold has a porous surface through which cells can proliferate and invade, and the stem cells of the present invention are easily attached onto the porous collagen scaffold and cultured. It was propagated, and even when the electric stimulation was applied, it could be stably attached and cultured and propagated (FIG. 2).

Example  3: Real time Reverse transcriptase -Polymerase chain reaction ( real time reverse  transcriptase-polymerase chain reaction ; real time RT - PCR Gene expression analysis using

First, gene expression according to three-dimensional cell culture was compared. Real time RT-PCR was performed by a known method.

As a result, as shown in Figure 3, it was confirmed that the gene expression is significantly increased in the 3D culture attached to the collagen scaffold culture than 2D planar culture. Specifically, 3D culture compared to 2D culture, IGF-1 (insulin-like growth factor-1) 74 times, BMP2 (bone morphogenetic protein 2) 59.4 times, CXCL2 (chemokine CXC motif ligand 2) 13.1 times, IL- 8 (interleukin-8) was increased by 149 times and vascular endothelial growth factor (VEGF) 1.2 times (Fig. 3).

Furthermore, in order to confirm the effect of electrical stimulation on gene expression in 3D culture, gene expression levels were measured on days 2 and 4 of 3D culture in the absence and presence of electrical stimulation, and the results are shown in FIG. 4.

As a result, it was confirmed that gene expression of saint factor related bone regeneration was increased by electric stimulation although there was a difference in degree and timing. That is, the IGF-1 gene increased 1.6 fold on the second day of culture, and the BMP2, VEGF, TGF-β and bFGF genes increased 4.2 fold, 2.3 fold (data not shown), 2.3 fold and 2.5 fold on the fourth day, respectively. On the other hand, the expression of the cytokines CXCL2 and IL-8 genes, which contribute to stem cell and early immune response-related cell migration, increased by 2.8 and 2.5 times, respectively, on day 4 (FIG. 4).

Example  4: ELISA ( enzyme - linked immunosorbent assay Protein expression analysis

From Example 3, it was confirmed that the expression level of bone regeneration-related growth factors and stem cells and early immune response-related cell migration-promoting cytokine genes were increased in 3D culture and / or by electrical stimulation. Therefore, the expression levels of the proteins were compared and analyzed using ELISA.

Specifically, after culturing the cells for 3 days, the culture medium was collected and vascular endothelial growth factor (VEGF) required for bone formation in the culture medium, basic fibroblast growth factor (bFGF), which plays an important role in cell proliferation and development, and The expression level of IL-8 (interleukin-8), a cytokine involved in stem cell migration, was confirmed.

As a result, VEGF, which plays an important role in angiogenesis, was present at a concentration 19 times higher in 3D culture using collagen scaffold than in 2D planar culture, and when electrostimulated (EC) was applied in 2D and 3D culture, Stimulation increased by 1.37 and 1.13 fold, respectively. On the other hand, bFGF was present at about 2 times higher concentration in 3D culture conditions than in 2D culture, but the effect by electric stimulation was observed in 2D culture, but not in 3D culture (data not shown). Finally, it was confirmed that cytokine IL-8 was secreted at a concentration of 26.5 times higher than that of 2D planar culture by 3D stereoculture alone, and increased by 1.38 and 2.02 times, respectively, by electric stimulation (FIG. 5).

In conclusion, the culture medium containing high concentration of growth factors and cytokines (IGF-1, VEGF, BMP2, bFGF, CXCL2, IL-8) was obtained by 3D stereoculture using collagen scaffold, and further electrical stimulation. As a result, the culture medium with increased expression of VEGF, bFGF and IL-8 could be obtained.

Example  5: Concentration of Stem Cell Culture and Evaluation of Activity of Concentrated Culture

The culture solution obtained by culturing the stem cells by the method of Example 2 was concentrated using a protein concentration filter (sartorius, vivaspin 2). The filter can be used to highly concentrate 1.5 ml of culture to 30 μl. Table 1 summarizes the total volume of culture in 2D and 3D cultures and the time it takes to concentrate using a column.

As a result, when using the 3D culture conditions in which cells are attached to the collagen scaffold and cultured, only 3 ml of the culture medium is used for 1 × 10 ⁶ cell culture, whereas the same number is used for the planar (2D) culture in the culture dish. 14 ml of culture was required to incubate cells, and therefore the concentration time also took 3.5 times or more. These results support that the 3D culture method of the present invention can concentrate the culture solution in a small amount of medium and a short time.

Through Examples 3 and 4, it was confirmed that growth factors and cytokines were present in higher concentrations in the culture medium obtained in the 3D culture than in the culture medium obtained from the 2D culture, and expected due to the volume difference of the culture medium used for the same number of cell cultures. In consideration of the difference in concentration time, the concentration of the culture solution using the protein concentration filter was performed only for the 3D culture solution.

Table 2 lists the concentrations of the protein measured in and outside the filter before and after filtration to determine the concentration. Although there is a loss within about 10%, the growth factor and cytokines with increased expression in most cultures are concentrated without loss to the outside of the filter due to the pre-filtration and the concentration of the protein concentrated inside the filter after filtration. It was confirmed that this was successful.

The concentrated culture solution was aliquoted and stored frozen and then thawed as needed in subsequent experiments.

Example  6: Effect of Stem Cell Concentrate on Cell Proliferation

In order to confirm the effect on the cytotoxicity and cell proliferation of the concentrated culture medium, the culture medium obtained by culturing with control (con) and electric stimulation (EC) was concentrated and mixed with general culture medium at a ratio of 1, 2.5 and 5%. Incubated for 3 days and confirmed using a cell counting kit-8 (CCK-8).

As shown in FIG. 7, it was confirmed that the experimental groups incubated with the concentrated culture mixture showed increased absorbance than the control group, so that cytotoxicity due to the concentrated culture was not shown. Furthermore, cell proliferation of 1.17 and 1.23 times higher was observed in the experimental group to which 1% and 2.5% concentrated culture medium was added, respectively. On the other hand, the effect of the addition of the concentrate of the culture obtained by stimulation with electric stimulation on the cell proliferation was similar.

Example  7: Effect of Stem Cell Concentrate on Differentiation into Osteocytes

In order to confirm the effect of the stem cell enriched culture of the present invention on the differentiation of stem cells into osteoblasts, the ALP (alkaline phosphatase) activity and the degree of alizarin red S stain were confirmed. ALP is an enzyme that is present in many parts of the body, especially bones and livers, and is present in osteoblasts, osteoblasts, and Alizarin Red S has a property of binding to metal ions. mineralization). Therefore, the degree of bone formation can be determined to the extent of the ALP activity and alizarin red S staining.

First, stem cells were treated with a control (con) and osteoblast differentiation conditions (dex). The control group was cultured in a medium to which ascorbic acid and β-glycerophosphate were added, and the osteoblast differentiation condition of the positive control group was cultured by adding dexamethasone (dexametasone) to the control medium. The experimental group was cultured using a medium containing 1% or 5% of the culture solution obtained by 3D culture in the presence / absence of electric stimulation.

8 shows the results of ALP activity measurement of the control group and the experimental group. As a result, when the medium containing 1% or 5% of the concentrated culture medium was increased compared to the osteoblast differentiation condition in which only dexamethasone was added to the control medium, the differentiation capacity to osteoblasts increased by 16 to 37%. However, no further increase due to electrical stimulation was observed.

9 shows the results of alizarin red S staining. First, using a culture medium containing a positive control group (Dex) and a concentrated culture solution except a negative control group (con) using a common culture medium (3D-con 1%, 3D-EC 1%, 3D-con 5% and 3D-EC) 5%) was used to induce differentiation into osteocytes, and the cells were fixed with 10% formalin on day 21, and then fixed solution was removed with distilled water. Then, the Alizarin Red S staining solution was added and reacted for 5 to 10 minutes, and then the dyeing solution was removed with distilled water and left for 15 minutes in PBS. Stem cells differentiated into stained osteocytes were photographed and visually decolorized to determine the degree of differentiation into osteoblasts. After reacting with de-stain solution for 15 minutes, ELISA was performed at 595 nm with the obtained solution to quantitatively compare the degree of alizarin red S staining. The concentration of stained Alizarin Red S, quantitatively indicated, was consistent with the results of the previous visual evaluation. That is, in the experimental group (3D-con 1%) differentiated into a culture medium containing 1% of the concentrated control medium obtained by 3D culture without the positive control group (Dex) and the electrical stimulation (3D-con 1%), but the degree of differentiation into osteoblasts was 3D culture Experimental group (3D-EC 1%, 3D-EC 5%) and the culture group containing 5% of the concentrated culture medium obtained by 3D culture without electric stimulation. In (3D-con 5%), it was confirmed that differentiation into osteoblasts was actively made.

Example  8: Concentration of Concentrated Broth Using Animal Experiment Model Osteogenesis  Activity evaluation

The concentrated and cryopreserved cultures were thawed and transplanted into a rat cranial defect model to confirm the degree of bone formation activity in vivo. After cranial tube defects with a diameter of 5 mm were formed by using a trephine bar on the left and right sides of the rat's skull, adult stem cells, concentrated culture medium obtained by 3D culture (media-C), and electrical stimulation were obtained by 3D culture. A concentrated culture medium (media-EC) was loaded into the collagen scaffold, a biodegradable solid support, implanted into the damaged area, and killed at 2 and 4 weeks to confirm the degree of bone regeneration. 10 shows a cranial tube defect modeling process and transplantation process.

Example  9: Determination of Inflammatory Response in Concentrated Cultures

In order to confirm whether the inflammatory reaction is induced by the transplanted concentrated culture medium, two weeks after transplantation, the transplanted rats were killed to prepare tissue sections, and the inflammatory cells were identified by H & E staining.

As a result, as shown in FIG. 11, cells that appeared to be inflammatory cells in the group transplanted with the stem cells (Cell) and the concentrated culture medium (Media-Con and Media-EC) transplanted groups were not identified. That is, the inflammatory response due to the transplanted stem cells and the concentrated culture medium was not induced. However, the transplanted collagen was found to still have some remaining.

Example  10: stem cell migration promoting effect of concentrated culture

In the same manner as in Example 9, two defects were prepared in the rat skull, and on one side, a scaffold loaded with a general culture solution and the other, a scaffold loaded with a concentrated culture obtained by 3D culture was implanted. The day after implantation, 5 × 10 ⁶ human bone marrow-derived mesenchymal stem cells (hBMSC) labeled with Dil, a fluorescent dye, were injected through the tail vein of the rat and perfused after 4 days. The positive control group was prepared using SDF-1 (50 μg / ml), known as a stem cell migration promoter, instead of the concentrated culture medium in the same experimental animal model. Stem cell migration was confirmed by confocal microscopy after the rats were killed and the scaffolds were recovered and frozen.

As a result, as shown in FIG. 12, in the control group injected with the normal culture solution, the fluorescence of the human bone marrow-derived stem cells injected from the tail could not be observed, but in the positive control group and the experimental group. In conclusion, it was confirmed that the concentrated culture medium of the stem cells contained a large amount of a substance having a stem cell migration promoting effect and promoted the stem cell migration similarly to the injection of the stem cell migration promoting material SDF-1. By grafting only the culture medium without transplanting stem cells that can directly differentiate into tissues, it is shown that the regeneration of autologous stem cells to the wound site can be promoted to induce tissue regeneration.

Example  11: concentrated culture Bone regeneration  effect

<11-1> micro CT Using Osteogenesis  Quantitative Analysis of Degrees

Three-dimensional micro CT imaging was used to quantitatively analyze the degree of bone formation from the scaffold loaded with the stem cells or the concentrated culture medium (FIG. 13). As a control, stem cells were attached to the 3D collagen scaffold and cultured for 4 days, and then the transplanted Cell group was used. The experimental groups (Media-C and Media-EC) were transplanted with the concentrated culture solution loaded into the same scaffold without cells, which showed 219 and 277% increased bone formation volume (BV) than the Cell group, respectively. It was. The trabecular bone number also increased by 202 and 270%, but the trabecular separation and thickness formed similarly in all groups. The synergistic effect of electrostimulation did not occur in the concentrated culture medium obtained by stimulation with electric stimulation, but the experimental group transplanted with the concentrated culture medium obtained by 3D culture regardless of the presence or absence of electrostimulation showed superior bone regeneration effect than the control group with stem cell transplantation. Indicated. Figure 14 shows a three-dimensional image obtained by micro CT imaging. Four weeks after loading stem cells or concentrated culture medium into bone defect model, newly formed bone (yellow) morphology was produced as an image. New bone tissue (yellow) was observed more in the experimental group (Media-C and Media-EC) transplanted with concentrated culture than in the control group (Cell) transplanted with stem cells, which is consistent with the results of the previous micro CT analysis.

<11-2> Histological staining Osteogenesis  evaluation

In order to analyze the new bone tissue from the scaffold loaded with stem cells or concentrated culture, it was confirmed histologically using Masson trichrome stain. The rat cranial defect model prepared by the method described in Example 8 was implanted with a scaffold loaded with stem cells or concentrated culture and stained 4 weeks later. As shown in Figure 15, the new bone tissue in the experimental group (Media-C and Media-EC) transplanted the concentrated culture medium than the control cell (Cell) transplanted stem cells in accordance with the micro CT quantitative analysis results and three-dimensional image More was observed. Specifically, in the group transplanted with stem cells, little new bone tissue was identified, and only collagen scaffolds transplanted together with a solid support were observed. On the other hand, when the concentrated culture solution was transplanted, a considerable amount of new bone tissue was observed, and in proportion to this, collagen scaffolds were degraded and decreased.

Claims (14)

Method for producing a culture medium comprising a growth factor or cytokine comprising the step of attaching the adult stem cells to the scaffold three-dimensional culture.
The method of claim 1,
The adult stem cells are bone marrow-derived mesenchymal stem cells.
The method of claim 1,
Collecting and concentrating the culture.
The method of claim 1,
Additionally comprising the step of culturing with electrical stimulation.
5. The method of claim 4,
Collecting and concentrating the culture.
The method of claim 1,
Wherein said scaffold is a collagen scaffold.
The method of claim 1,
The growth factor is IGF-1 (insulin-like growth factor-1), TGF-β1 (transforming growth factor-beta 1), bFGF (basic fibroblast growth factor), BMP2 (bone morphogenetic protein 2) or vascular endothelial growth method).
The method of claim 1,
Wherein said cytokine is chemokine CXC motif ligand 2 (CXCL2) or interleukin-8 (IL-8).
A concentrated culture medium comprising a growth factor or cytokine prepared by the method of any one of claims 1 to 8.
The composition for bone tissue regeneration comprising the concentrated culture of claim 9 as an active ingredient.
(a) preparing a culture solution by the method of claim 1; And
(b) loading the prepared culture solution into a transplant scaffold;
A method for producing a bone tissue regeneration implant comprising a scaffold for transplantation loaded with a culture solution.
12. The method of claim 11,
Wherein said implant scaffold is a biodegradable material.
The method of claim 12,
The biodegradable material is collagen manufacturing method.
Implant for bone tissue regeneration comprising a scaffold for transplantation loaded with the culture medium prepared by the method of claim 11.

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