WO2016163828A1 - Polymer support for increasing binding between cells, and method for culturing cells by using same - Google Patents

Abstract

The present invention relates to a polymer cell support for inducing binding between cells. The polymer cell support of the present invention allows culture cell-cell adhesion to be induced, thereby providing a composition for inducing cell differentiation, a composition for cell transplantation, or the like.

Description

Polymer support for enhancing cell-to-cell binding and cell culture method using the same

The present invention was made by the task number NRF-2013R1A2A2A03010055 under the support of the Ministry of Science, ICT and Future Planning, the research management specialized organization of the project is the Korea Research Foundation, the research project name is "medium-level researcher support project," Tissue regeneration using sensitized microgels ”, the host institution is Hanyang University Industry-Academic Cooperation Group, and the research period is from 06.01.

This patent application is filed with the Korean Patent Application No. 10-2015-0050380 filed with the Korean Patent Office on April 9, 2015 and the Korean Patent Application No. 10-2016-0043529 filed with the Korean Patent Office on April 8, 2016. Priority is claimed, the disclosures of which are incorporated herein by reference.

The present invention relates to a polymeric cell support that induces binding between cells.

In tissue engineering and regenerative medicine, cell therapy is commonly used to restore damaged tissue and regeneration. Currently, various treatments using stem cells have been carried out. However, simple injection of cells has low engraftment rate and difficulty in controlling differentiation, and many studies have been conducted to maximize such effects. In addition, in the use of stem cells as therapeutic agents, various methods have been developed to maximize the engraftment rate and differentiation capacity of cells in vivo. It is essential to use a support for effective cell delivery and tissue regeneration, and recently, cell-bound support using polymers has been developed. Most of the polymer scaffolds are used to fix the cell binding material in order to increase the cell binding capacity, or to fix the growth factor and growth factor-derived peptides to induce differentiation of cells, but have not reached a satisfactory level.

Such conventional polymer scaffolds are focused on enhancing the binding force between cells and extra cellular matrix. However, none of the cells in vivo exist only by the binding between the extracellular matrix, but exists in an environment that binds to various other cells. Therefore, there is a need for the development of a support that can provide cell-to-cell binding in culturing cells in vitro and in vivo .

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The inventors have made intensive research efforts to develop polymeric cell scaffolds that provide cell-cell binding. As a result, in the case of using a polymer support conjugated with a cadherin-attached oligopeptide, not only the cell-supporting bond is formed, but also the cell-cell binding is induced to be useful for cell culture and stem cell differentiation. By clarifying that the present invention has been completed, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a polymeric cell support for cell population formation.

Another object of the present invention to provide a cell colonization culture method.

Still another object of the present invention is to provide a composition for inducing stem cell differentiation.

Another object of the present invention to provide a stem cell culture method for differentiation into chondrocytes.

Another object of the present invention to provide a cell composition for implantation in the body.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the invention provides a polymeric cell support for forming a cell population comprising:

(a) a biocompatible polymeric backbone;

(b) Cadherin adherent oligopeptide bound to the polymer backbone.

The inventors have made intensive research efforts to develop polymeric cell scaffolds that provide cell-cell binding. As a result, in the case of using a polymer support conjugated with a cadherin-attached oligopeptide, not only the cell-supporting bond is formed, but also the cell-cell binding is induced to be useful for cell culture and stem cell differentiation. It was found possible.

Conventional cell culture polymer supports provide binding between cells and supports, but do not provide the effect of inducing binding between cells. However, the polymer cell support of the present invention includes a cDHERIN-attached oligopeptide, thereby forming a bond with CADHERIN protein on the surface of the cultured cell, and inducing cell-cell binding, thereby enabling clustering of cultured cells. There is a characteristic. "Cardherin protein" is a type 1 transmembrane protein, a protein that plays an important role in forming interhesion.

As used herein, the term “biocompatible polymer backbone” refers to a structure made of a biocompatible polymer that has affinity upon contact with a cell and does not exhibit a rejection reaction, and is a material of a structure such as a scaffold for conventional cell culture. Various materials known to be used may be used without limitation. However, it is necessary to include a functional group for binding a caherin adherent peptide, or to be surface modified to include such a functional group. The polymer backbone may be prepared in various forms by various methods, and is not particularly limited. The polymer backbone of the present invention may be prepared, for example, in the form of microspheres, and for this, a water-in-oil emulsion method may be used. See the embodiments on this specification for specific methods.

As used herein, the term “cadherin adherent oligopeptide” refers to an oligopeptide sequence capable of forming a bond with a cadherin protein of a cultured cell, wherein the oligopeptide sequence forms a bond with a cadherin protein on a cell. It will be obviously understood that the effects of the present invention are significantly exerted only if so. Identification of oligopeptides that form bonds with Cadherin proteins can be carried out through a variety of methods known in the art, such as two-hybrid analysis or three-hybrid analysis (US Pat. No. 5,283,317; Zervos et. al., Cell 72, 223-232, 1993; Madura et al., J. Biol . Chem . 268, 12046-12054, 1993; Bartel et al., BioTechniques 14, 920-924, 1993; Iwabuchi et al., Oncogene 8, 1693-1696, 1993; and WO 94/10300). In this case, an oligopeptide that binds to the cadrerin protein can be screened by using the cadrerin protein as a bait protein. Two-hybrid systems are based on the modular nature of the transcription factors composed of cleavable DNA-binding and activation domains. For simplicity, this assay uses two DNA constructs. For example, in one construct, a cadrine-encoding polynucleotide is fused to a DNA binding domain-encoding polynucleotide of a known transcription factor (eg, GAL-4). In another construct, a DNA sequence encoding an oligopeptide (“prey” or “test material”) of interest is fused to a polynucleotide encoding the activation domain of the known transcription factor. If bait and prey interact in in vivo to form a complex, the DNA-binding and activation domains of the transcription factors are contiguous, which triggers transcription of the reporter gene (eg, luciferase). As a result, the expression of the reporter gene can be detected, which indicates that the prey can bind with the cadrerin protein, which means that the prey can be used as the cadrerin adherent oligopeptide of the present invention.

In one embodiment of the present invention, the bond between the polymer backbone and the catherine attachable oligopeptide of the present invention is an amide bond formed between the biocompatible polymer backbone described above and the catherine attachable oligopeptide described above. Cadherin adherent oligopeptides can be associated with the polymeric backbone using N-terminal or C-terminal amino or carboxyl groups. At this time, the polymer backbone of the present invention is required to include a functional group capable of forming an amide bond with a cadmin attached oligopeptide.

In one embodiment of the present invention, the biocompatible polymer backbone of the present invention comprises a carboxyl group, hydroxyl group or amino group for forming the amide bond. Specifically, the polymer backbone of the present invention not only includes a carboxyl group or a hydroxyl group capable of binding to the amino group of the caherin-adhering oligopeptide, or an amino group capable of bonding to the carboxyl group of the caherin-adhering oligopeptide, It is understood to include surface modified to include carboxyl, hydroxyl, or amino groups. Although amide bond formation between carboxyl group and amino group is more generally known, amide bond formation between hydroxyl group and amino group is also known by using an organometallic catalyst (Kim Ki-Chul et al., Such as amide using an organometallic catalyst from an alcohol and an amine). The development of bond formation reactions, the chemical world, 2013. 3), through the various known methods can be introduced caherin adhesion oligopeptide to the polymer backbone of the present invention. Specifically, for example, the EDC / NHS reaction used in one embodiment of the present invention can be used. On the other hand, the binding between the polymer backbone of the present invention and the cadherin oligopeptide may be made through a peptide linker.

In one embodiment of the present invention, the polymer backbone of the present invention may include a carboxyl group or hydroxyl group, or a surface modified to include a carboxyl group or hydroxyl group. In the present embodiment, the polymer backbone of the present invention may form an amide bond with the N-terminal amino group of the cadherin-attached oligopeptide, or form a bond with the C-terminal carboxyl group of the cadherin-attached oligopeptide via a linker. It is possible. Linkers capable of forming bonds between carboxyl groups in the present invention are specifically, for example, diamine, divinylsulfone, 1,4-butanediol, diglycidyl ether (BDDE) and glutaraldehyde, Carbodiimide, hydroxysuccinimide, imidoester, maleimide, haloacetyl, disulfide, hydroazide, or alkoxyamine may be used, but is not limited thereto.

In one embodiment of the present invention, the polymeric backbone of the present invention is any one or more selected from the group consisting of alginic acid polymers, chitosan polymers and hyaluronic acid polymers. The “alginic acid polymer” of the present invention is a polymer polymer represented by Formula 1, and is a polymer suitable for forming a bond with a cadmin adherent oligopeptide of the present invention including a carboxyl group.

[Formula 1]

In addition, the "chitosan polymer" of the present invention is a polymer polymer represented by the formula (2), and includes a hydroxyl group and an amino group, and is a polymer suitable for forming a bond with the cardherin adherent oligopeptide of the present invention.

[Formula 2]

In addition, the "hyaluronic acid polymer" of the present invention is a polymer represented by the formula (3), and includes a hydroxyl group and a carboxyl group, and is a polymer suitable for forming a bond with the cardherin adhesion oligopeptide of the present invention.

[Formula 3]

In addition, a polymer polymer surface-modified to include a carboxyl group, hydroxyl group or amino group can be suitably used. As for the method of surface modification, a method described in the prior art can be used without limitation, and specifically, introducing a carboxyl group to the surface of the polymer polymer by graft using acrylic acid (AAc), for example, It is possible.

In one embodiment of the present invention, the caherin adherent oligopeptide of the present invention is an LRP5 peptide consisting of SEQ ID NO: 1 or HAV peptide consisting of SEQ ID NO: 2. "LRP5 peptide" of the present invention is a sequence of the first part of the sequence list as a part of the amino acid sequence of the low-density lipoprotein receptor-related protein 5 (LRP5) that binds effectively to the cardherin protein. "HAV peptide" of the present invention is a peptide consisting of the amino acid sequence of a specific site of the cardinin protein directly participating in the tight junction (tight junction) of the cell consisting of the second sequence of the sequence listing. The inventors anticipated that the effect of the present invention would be caused by caherin adhesion, independent of sequence specificity, and demonstrated this using two sequences of caherin attachment peptides that were not related to each other.

In one embodiment of the present invention, the LRP5 peptide or HAV peptide of the present invention forms a bond with the cardherin protein of the cell. Cadherin protein is a protein that plays an important role of directly participating in the intercellular adhesion (adhesion), as described above, the present inventors target the cadherin protein oligopeptides, specifically examples For example, using the LRP5 peptide and / or HAV peptide, a polymer cell support capable of forming a cell population was prepared.

In one embodiment of the present invention, the cell support of the present invention further comprises (c) an integrin adherent oligopeptide bound to the above-described polymer backbone. The "integrin" of the present invention is a transmembrane receptor that acts as a bridge of cell-cell and extracellular matrix (ECM) interactions. As used herein, the term "integrin adherent oligopeptide" refers to a peptide strand consisting of approximately 2-20 amino acids that form a bond with the aforementioned integrins. In the case of culturing cells using a cell support including integrin-attached oligopeptides in addition to the cadherin-attached oligopeptides described above in the biocompatible polymer backbone of the present invention, the cell population is more compact in spherical form. It was confirmed that it was formed.

In one embodiment of the invention, the integrin adherent oligopeptides of the invention are RGD, PSHRN, FHRRIKA, YIGSR, KGD, RHD, NGR, SDGR, KQAGDV, LDV, DGEA, YGYYGDALR, FYFDLR, DALR, DLR, RLD, KRLDGS, IDA, IDAPS and REDV. PSHRN (Benoit and Anseth, 2005), FHRRIKA (Rezania and Healy, 1999), YIGSR (Massia and Hubbell, 1990), KGD (Plow et al, 1985; Scarborough et al 1993), RHD (Ghiso et al 1992, Saporito-Irwin & van Nostrand 1995), NGR, SDGR (Yamada & Kennedy 1987), KQAGDV (Kloczewiak et al, 1984; Lam et al 1987), LDV (Guan & Hynes 1990, Mold et al 1990), DGEA (Staatz et al 1991), YGYYGDALR, FYFDLR (Underwood et al 1995), DALR, DLR, RLD (Altieri et al, 1993; Koivunen et al, 1995), KRLDGS (Alfieri et al 1993), LDV, IDA, IDAPS and REDV (Mould) et al, 1990; Mold & Humphries, 1991) are oligopeptides that bind to integrins, such as RGD peptides.

According to another aspect of the present invention, the present invention provides a cell colonization culture method comprising the step of culturing the cells in contact with the cell support described above.

For conventional cell culture, it is common to use a feeder cell layer for cell attachment. However, in the case of using the polymer cell support of the present invention, it acts like a feeder cell layer, and not only forms a cell-supporter bond, but also induces a cell-cell bond to form cells in a cell population. It can be cultured. There is no particular limitation on the cells that can be cultured by the method of the present invention.

Cell culture of the present invention can be made in conventionally used cell culture medium. The cell culture medium may be appropriately selected and used depending on the type of cultured cell and the purpose of the culture. The basal medium constituting the medium of the present invention is a conventional medium in the art, such as DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)), Eagle's MEM [Eagle's minimum essensial medium, Eagle, H. Science 130: 142 (1959)], α-MEM [Stanner, CP et al., NAT. New Biol . 230: 52 (1971)], Iscove's MEM [Iscove, N. et al., J. Exp . Med . 147: 923 (1978), 199 medium [Morgan et al., P roc . Soc. Exp . BioMed . 73: 1 (1950), CMRL 1066, RPMI 1640 [Moore et al., J. Amer . Med . Assoc . 199: 519 (1967)], F 12 [Ham, Pro. Natl. Acad . Sci . USA 53: 288 (1965)], F 10 [Ham, RG Exp . Cell Res. 29: 515 (1963)], a mixture of DMEM and F12 [Barnes, D. et al., Anal. Biochem . 102: 225 (1980), Way-mouth's MB752 / 1 [Waymouth, CJ Natl . Cancer Inst . 22: 1003 (1959)], Dulbecco's modified device Cove medium (Iscove's modified Dulbecco's medium), device Cove modified Fischer medium or device Cove modified Eagle's medium, McCoy's 5A [McCoy, TA, et al, Pro. soc. Exp . Bio. Med . 100: 115 (1959)], MCDB's series (Ham, RG et al., In Vitro 14: 11 (1978)), AIM-V medium and its modified medium. A detailed description of the medium can be found in R. Ian Freshney, Culture of Animal Cells, A Manual of Basic Technique, Alan R. Liss, Inc., New York, the contents of which are incorporated herein by reference. In addition, the medium of the present invention may additionally include antibiotics, and may include, but are not limited to, for example, penicillin, streptomycin, gentamycin, neomycin, polymyxin or amphotericin B.

According to another aspect of the invention, the present invention provides a composition for inducing stem cell differentiation comprising the above-described cell support. The "stem cells" of the present invention are not particularly limited, and all of the cells having the characteristics of stem cells, that is, undifferentiated, indefinite proliferation and differentiation into specific cells are cells that can be applied to the present invention. Stem cells to which the present invention is applied are largely divided into pluripotent stem cells and multipotent stem cells, including embryonic stem cells (ES) and embryonic germ cells (EG). Embryonic stem cells are derived from the internal cell mass (ICM) of the blastocyst, and embryonic germ cells are derived from primordial germ cells of the 5-10 week old gonadal ridge. Pluripotent stem cells, on the other hand, are found in embryonic, fetal and adult tissues, including adult stem cells. Pluripotent stem cells proliferate indefinitely in vitro and have the ability to differentiate into a variety of cells derived from all three embryonic layers (ectoderm, mesoderm and endoderm). Pluripotent stem cells, on the other hand, have the ability to differentiate into the specific tissue from which they originate, and the ability to autoreproduce is typically limited to the life of the organism. Sources of pluripotent stem cells are all tissue types and are primarily isolated from bone marrow, blood, liver, skin, intestine, pancreas, brain, skeletal muscle and pulp.

The present invention is characterized by inducing colonization regardless of the concentration of cultured cells using the above-described cell support, and promoting cell differentiation by cell-cell binding formed at this time, and other culture conditions are greatly limited. Instead, the cells can be appropriately selected and adjusted according to the cultured cells and the culture purpose. The composition of the present invention may include a conventionally known stem cell differentiation condition medium, the differentiation condition medium can be used by appropriately selecting a conventionally known medium according to the type of cells to be obtained as a result of differentiation.

According to another aspect of the present invention, the present invention comprises the step of contacting the stem cell differentiation induction composition and the stem cells described above and culturing in chondrocyte differentiation conditions medium, stem cell culture method for differentiation into chondrocytes To provide.

The term "chondrocyte differentiation condition medium" of the present invention refers to a medium under conditions that induce differentiation of stem cells into chondrocytes, and as a representative example, insulin, transferrin, selenium acid, woo supplemented with growth factors Serum-deficient media (Johnstone et al., 1998) comprising serum albumin, linoleic acid, pyruvate, ascorbate and / or dexamethasone, and DMEM / F12, dexamethasone, L used in one embodiment of the present invention. A medium containing ascorbic acid, insulin and / or TGF-β1 may be used, but is not limited thereto.

In the stem cell culture method for differentiation into chondrocytes of the present invention, in particular, in the case of using a cell support further comprising an integrin-attached oligopeptide, as well as a cadrerin-attached oligopeptide, a cadherin- or integrin-attached Gene expression of chondrocyte differentiation factor was significantly increased as compared with the case of using a cell support including sex oligoptide alone. Therefore, preferably, stem cells may be cultured using a cell support including both a caherin-adhering oligopeptide and an integrin-adhering oligopeptide, and more effectively differentiated into chondrocytes.

According to another aspect of the present invention, the present invention provides a composition for transplantation in vivo cells comprising (a) the cell support described above, (b) cells for transplantation in vivo bound to the cell support.

Cells for transplantation of the present invention are specifically, for example, chondrocytes, myoblasts, hepatocytes, osteoblasts, embryonic stem cells, embryonic germ cells (embryonic) germ cells, adult stem cells, mesenchymal stem cells, neural stem cells, vascular endothelial stem cells, hematopoietic stem cells, liver stem cells, heart stem cells, pancreatic stem cells, endothelial progenitors, growth Outgrowth endothelial cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, satellite cells, intestinal epithelial cells, smooth muscle cells and fibers There are blasts, but not limited thereto. The cell composition for implantation of the body of the present invention can be injected at a desired location using, for example, injection or surgical methods.

In one embodiment of the invention, the cells for transplantation in the body of the invention are stem cells. The stem cells are as described above in the present specification, and may be used in a conventionally known use mode for stem cell therapy for treating diseases by introducing stem cells into the body.

In one embodiment of the present invention, the cells for transplantation in the body of the present invention are chondrocytes differentiated from stem cells.

The composition of the present invention may additionally include a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier included in the therapeutic composition of the present invention is commonly used in the formulation, lactose, dextrose, sucrose, Sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzo 8, talc, magnesium stearate, mineral oil, and the like. Therapeutic compositions of the present invention may further comprise lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components. Pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The compositions of the present invention can be administered parenterally, and local injection is the most preferred method of administration.

Suitable dosages of the compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and response to reaction. have. On the other hand, the dosage of the composition is preferably 1 times ^{1 x 10 5 - 5 x 10} 8 cells / ml and can be adjusted by the amount required.

The compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. The formulations may then be in the form of solutions, suspensions or emulsions in oil or aqueous media, and may further comprise dispersants or stabilizers.

The compositions of the present invention allow for excellent formation of intercellular networking to fully exert the function of the cells to be transplanted, thereby actually exhibiting improved therapeutic efficacy in diseased animals.

Intracellular graft cell compositions of the present invention can be used for the treatment of cartilage-injury diseases, specifically, for example, osteoarthritis, rheumatoid arthritis, meniscus cartilage injury (Meniscus Injury), It can be used for the treatment of Costoschondritis, Relapsing polychondritis, and Chondrosacoma.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a polymer cell support for forming cell populations.

(b) The present invention provides a cell colonization culture method.

(c) The present invention provides a composition for inducing stem cell differentiation.

(d) The present invention provides a stem cell culture method for differentiation into chondrocytes.

(e) The present invention provides a cell composition for implantation in the body.

(f) The polymer cell support of the present invention can induce cell-cell conjugation to form cell populations even when small concentrations of cells are used in cell culture.

(g) Using the composition for inducing stem cell differentiation of the present invention and the stem cell culture method, it is possible to effectively differentiate the stem cells into desired cells.

Figure 1 schematically shows the attachment process of the cells using the support to which the cell adhesion peptide of the present invention is immobilized.

Figure 2 shows the process and predicted structure of the cell-adhesive peptide of the present invention binds to the polymer structure of the polymer support.

Figure 3 shows the results of confirming that the cell-adhesive peptide is bound to the polymer support as a result of the IR analysis of the polymer support to which the cell-adhesive peptide is bound.

Figure 4 shows the results of measuring the physical properties of the polymer support bound to the cell-adhesive peptide of the present invention.

Figure 5 shows the results of observing the phenotype of each cell by attaching a variety of cells to the polymer support to which the cell adhesion peptide of the present invention is bound.

Figure 6 shows the results of observing the binding force between cells of the cells attached to the polymer support to which the cell-adhesive peptide of the present invention is bound.

Figure 7 shows the effect of the cell-adhesive peptides of the present invention on the phenotype of stem cells, and shows the results confirmed that they are attached by a specific binding force between cells.

Figure 8 shows the results of observing the phenotype of the stem cells and the binding force between the cells when differentiating stem cells into chondrocytes using a low concentration of the cell number of the cell adhesion peptide conjugated polymer support of the present invention.

Figure 9 shows the results of analyzing the differentiation capacity when differentiating stem cells to chondrocytes using low concentration of the cell-adhesive peptide conjugated polymer support of the present invention.

Figure 10 shows the results of observing the phenotype and the intercellular binding force of the stem cells when the high concentration of stem cells to the chondrocytes using the polymer support conjugated to the cell-adhesive peptide of the present invention.

Figure 11 shows the results of analyzing the differentiation power when differentiation of high concentration of stem cells into chondrocytes using the polymer support to which the cell-adhesive peptide of the present invention is bound.

12 shows a method for forming a polymer microsphere and a process of binding a cell-adhesive peptide to the polymer microsphere.

Figure 13 shows the process of forming aggregates using the polymer microspheres and stem cells of the present invention.

Figure 14 shows the results of measuring the physical properties of the aggregates formed of the polymer microspheres and stem cells of the present invention.

Figure 15 shows the results of observing the phenotype of the stem cells in the aggregate formed of the polymer microspheres and stem cells of the present invention.

Figure 16 shows the results of observing the survival rate of stem cells in the aggregate formed of the polymer microspheres and stem cells of the present invention.

Figure 17 shows the results of histological evaluation (Alcian blue, serious red) to analyze the differentiation ability of stem cells into cartilage cells in aggregates formed of the polymer microspheres and stem cells of the present invention.

Figure 18 shows the results of analyzing the differentiation power when differentiating stem cells into chondrocytes in the aggregate formed of the polymer microspheres and stem cells of the present invention.

Figure 19 shows the results of analyzing the growth ability of the stem cells according to the concentration of the card herin peptide of the present invention.

Figure 20 shows the results of analyzing the phenotype and adhesion of stem cells according to the concentration of the cardinin peptide of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example 1 Preparation of Cellular Adhesion Oligopeptide-Assembled Polymer Support and Evaluation of Cell Adhesion

1.1 Preparation of Polymer Support

1.1.1 Selection of Peptides

In order to develop a polymer scaffold that can provide the cell-to-cell binding force in vivo, we tried to develop a scaffold incorporating LRP5 peptide (DSCPPSPATERSYFHLFPPPPSPCTDSS) that specifically binds to the cell's cadherin protein. (See FIG. 1).

1.1.2 Binding of Peptides

Synthesis was performed using alginic acid, a natural polymer, and the EDC / NHS reaction was used to bind the carboxyl group of alginic acid to the amine group of the cell-adhesive peptide. Alginic acid was dissolved in MES buffer at pH 6.5, and peptides were bound using EDC and NHS, and then the peptide-bound alginic acid solution was dialyzed with distilled water and NaCl for 3 days. Activated carbon was added to the aqueous solution of alginic acid to complete dialysis to remove impurities for 30 minutes, filtered through a 0.22 μl filter, and lyophilized (see FIG. 2).

1.1.3 Polymer Surface Analysis (Amino Acid Analysis)

According to the above embodiment, the amino acid analysis was performed on the polymer support to which the LRP5 peptide was attached to determine whether amino acids were efficiently bound (see Table 1).

TABLE 1

No amino acid was observed in the polymer support without the peptide attached, and the amino acid included in the peptide was observed in the polymer support to which the cell-adhesive LRP5 peptide was bound.

1.1.4 Polymer Surface Analysis (IR Analysis)

As a result of confirming the polymer support to which the peptide is not bound and the peptide to which the peptide is bound through IR analysis of the polymer support to which the LRP5 peptide is attached, the amide bond (1450 cm ^-1) on the peptide is bonded to the polymer support. ) Can be observed (see FIG. 3).

1.1.5 Properties of Polymer Support

As a result of measuring the complex elastic modulus of the LRP5 peptide-attached polymer support, it was observed that the polymer support to which the peptide was not bound had a slightly higher complex elastic modulus than the polymer support to which the peptide was attached. This is because the site where the peptide is bound in the structure of the alginic acid polymer coincides with the binding site for forming the alginic acid hydrogel, which is expected to decrease the complex elastic modulus caused by the binding of the peptide (see FIG. 4).

1.2 Evaluation of Adhesion of Various Cells

1.2.1 Evaluation of Adhesion of Various Cells and Observation of Cell Phenotypes Using Polymer Supports

In order to observe the cell adhesion ability and phenotype of the polymer support that provides the cell-to-cell binding, experiments were conducted using a polymer support in which RGD peptides alone provided the binding between the cell and the support. Adherence in various cells was evaluated using hematopoietic cells (HSC), osteoblasts (MC3T3), adult stem cells (D1 stem cells) and fibroblasts (NIH3T3).

Observation of cell adhesion and cell phenotype in the RGD peptide-bound polymer scaffold confirmed the binding to hematopoietic cells (HSC), osteoblasts (MC3T3), adult stem cells (D1 stem cells) and fibroblasts (NIH3T3). And evenly binding to single cells (see FIG. 5A).

In comparison with the above results, it was observed that each cell was combined to form a cell population in the polymer support to which the LRP5 peptides provided intercellular binding (see FIG. 5B).

1.2.2 Evaluation of Intercellular Binding Induction Capacity of Cadherin-Adhesive Oligopeptide-Bound Polymer Supports

Hepatoblasts (HSC), osteoblasts (MC3T3), adult stem cells (D1 stem cells) and fibroblasts (NIH3T3) were attached to the surface of the polymer support conjugated with the caherin-adhesive LRP5 peptide to induce intercellular binding. In addition, it was confirmed that each cell formed and attached to the cell population, and the intercellular binding protein, n-cadherin, was observed through immunostaining. (See FIG. 6).

Example 2: Evaluation of Chondrocyte Differentiation Ability of Stem Cells Using Polymer Supports Providing Cell-to-Cell Binding

2.1 Evaluation of Adhesion Capacity of Stem Cells

To evaluate the binding ability of the adult stem cells (D1 stem cell, CRL-12424, ATCC) using a polymer support that provides cell-to-cell binding, and to identify the phenotype of the cell and cell-cell binding. Experiments were carried out using a polymer scaffold in which RGD peptides alone were provided to provide a binding between the cell and the scaffold. The adult stem cells (D1 stem cell, CRL-12424, ATCC) used are adult stem cells derived from mouse bone marrow, and are very suitable for the study of differentiation into bone, cartilage and adipocytes.

In the polymer support to which neither LRP5 or RGD peptide is bound, the adhesion of stem cells is low, and thus a relatively small amount of cells are attached. Also, the expression of catenin protein, which confirms cell-cell binding ability, also appears. It could be confirmed that it does not (see Fig. 7). In contrast, in the polymer scaffold to which the RGD peptide and the LRP5 peptide were respectively bound, a large number of cells were attached. In the polymer scaffold to which the LRP5 peptide was bound, the cells and the cells bound to each other and formed a cell population. I could observe the appearance.

In order to confirm that the LRP5 peptide-bound polymer support provides cell-cell binding using Cadherin protein, the peptide was not bound as a result of binding to the support after inhibiting cell-cell binding by treating EGTA solution. Cell adhesion similar to that of the polymeric support could be observed (see FIG. 7). This is a result showing that the polymer support into which the catherine-adherent oligopeptide of the present invention is introduced provides cell-cell binding.

2.2 Evaluation of Chondrocyte Differentiation Capacity of Stem Cells

2.2.1 Evaluation of Cartilage Differentiation Capacity in Polymer Supports Using Low Concentration Cells

To evaluate the differentiation ability of stem cells into chondrocytes using a polymer support incorporating only RGD peptides that provide binding between cells and support and a polymer support incorporating LRP5 peptides providing binding between cells and cells, Were encapsulated and cultured under chondrocyte differentiation medium conditions (DMEM / F12, 10 nM dexamethasone, 50 μg / ml L-ascorbic acid, 5 μl / ml Insulin, 10 ng / ml TGF-beta1) for 2 weeks. At this time, the concentration of the stem cells used was incubated with the number of cells (1.0 x 10 ⁶ cells / ml) of a concentration lower than one tenth of the cell concentration used in the general three-dimensional cell culture method.

After culturing for 2 weeks, the cell phenotype was observed. In the polymer scaffold in which the RGD peptide was introduced, stem cells existed as single cells and immunostained beta-catenin, a protein that can confirm cell-to-cell binding. In contrast, it was confirmed that the binding was not performed, but in contrast, the polymer support in which the LRP5 peptide was introduced showed that the stem cells were clustered, and the beta-catenin protein expression was also markedly increased. (See FIG. 8).

In order to evaluate the chondrocyte differentiation ability of stem cells, gene expression levels of collagen type2 (collagen type2) and sox-9, which are chondrocyte differentiation factors, were measured. As a result, the RGD peptide was bound to the LRP5 peptide-coupled polymer scaffold. Compared with the polymer support, it was confirmed that a large amount of genes were expressed (see FIG. 9). In addition, this is in contrast to the result that cell culture and cell differentiation at low concentration are not performed well under the condition that the cells were cultured at the lower cell number of 1/10 than the concentration used for general three-dimensional cell culture. In other words, cell culture using a polymer support that provides cell-to-cell binding acts as a supporter that provides cell-to-cell binding even at low concentrations of cell culture. It was confirmed that it has a big impact.

2.2.2 Evaluation of Cartilage Differentiation Capacity in Polymer Supports Using High Concentration Cells

In order to evaluate the differentiation ability of chondrocytes used for three-dimensional culture of stem cells, experiments were carried out at a concentration of 1.0 x 10 ⁷ cells / ml, which is a commonly used cell concentration. The experiment was conducted using a polymer support in which a polymer support and an LRP5 peptide were bound.

Stem cells (D1 stem cell, CRL-) were cultured in chondrocyte differentiation medium (DMEM / F12, 10 nM dexamethasone, 50 μg / ml L-ascorbic acid, 5 μl / ml Insulin, 10 ng / ml TGF-beta1) for 2 weeks. 12424, ATCC) induced the differentiation of chondrocytes, and it was observed that RGD peptides were also forming a cell population in the polymer scaffold to which the RGD peptides provided the binding between the cell and the support. In the polymer scaffold to which the LRP5 peptide was bound, more cells were observed to form colonies (see FIG. 10).

As a result of measuring the gene expression levels of the chondrocyte differentiation factors collagen type 2 and sox-9, it was confirmed that the expression level of the gene was slightly increased in the polymer scaffold to which the LRP5 peptide was bound, but the experiment using the low concentration of the cells It was confirmed that it is not large in comparison with. This shows that as the concentration of the cells increases, even in the polymer scaffold to which the RGD peptide is bound, which does not provide the cell-to-cell binding, the cell-to-cell binding exists to form cell clusters. As the concentration of cells increases, LRP5 peptides do not appear to differ significantly from the polymer scaffold to which they are bound.

Example  3: cell Intercellular  Polymeric microspheres that provide a bond ( microsphere Cartilage cells of stem cells differentiation Ability evaluation

3.1 Preparation of Polymer Microspheres and Aggregation with Cells

3.1.1 Selection of Peptides

In order to develop polymer microspheres that can provide the cell-to-cell binding force, a specific site peptide (GGGGSHAVSS) (HAV peptide) of CHERIN itself, which directly participates in a cell tight junction to a polymer support, is selected or alone. By binding to the polymer support together with the RGD peptide, polymer microspheres capable of direct binding with the cardherin protein present in the cell membrane of the cultured cells were prepared.

3.1.2 Binding of Peptides

Microspheres were prepared using alginic acid, a natural polymer, and a water-in-oil emulsion was used to prepare alginic acid microspheres. Alginate polymer was dissolved in an organic solvent containing isooctane, SPAN 80, and Tween 80, and reacted in an aqueous solution of 20 wt% CaCl ₂ for one hour to prepare microspheres. Thereafter, an EDC / NHS reaction was used to bind the carboxyl group of the alginic acid microspheres to the amine group of the caherin-adhesive oligopeptide (HAV peptide). Alginic acid microspheres were dissolved in MES buffer at pH 6.5, peptides were bound using EDC and NHS, and the peptide-alginated aqueous alginic acid solution was dialyzed with distilled water and NaCl for 3 days. Alginate aqueous solution completed dialysis was added to activated carbon to remove impurities for 30 minutes, filtered through a 0.22 μm filter and lyophilized (see Fig. 12).

3.1.3 Evaluation of Stem Cell Growth Capacity by Cadherin Adhesion Peptide Concentration

In order to evaluate the stem cell growth ability according to the concentration of the caffeine adherent peptide, a hydrogel was prepared by introducing peptides of various concentrations (7, 14, 28, 70 μg / mg) into the alginic acid, to each hydrogel Stem cells were attached at a concentration of 2.0 × 10 ⁶ cells / ml and the experiment was performed for a total of 5 days. In order to measure the number of cells attached to the hydrogel, the hydrogel was removed with a solution containing EDTA, and after the polymer support was removed, the number of cells was measured by a hemocytometer to evaluate the growth ability of the stem cells for 5 days. . As a result of assessing growth ability by attaching stem cells to alginic acid to which the caherin-adhesive peptide was introduced, it was confirmed that the growth capacity was increased compared to alginic acid without the peptide. In addition, as the peptide concentration was increased in proportion to the increase, it was confirmed that the efficiency appears to be the highest when binding to more than 14 μg / mg concentration (reference: Figure 19). In addition, as a result of observing cells by attaching stem cells, it was confirmed that cell attachment was more active as the concentration of peptide increased, and that each cell was combined to form a cell population ( See FIG. 20).

3.1.4 Aggregation of Stem Cells and Polymer Microspheres

Agglomerates composed of stem cells and polymer microspheres were prepared by mixing the polymer microspheres and the stem cells having the binding force with the cells. The polymer microspheres were used at a concentration of 1.0 x 10 ⁸ / ml at 10 wt%, and the cells were 1.0 x 10. Aggregates were formed using a concentration of ⁷ / ml. Aggregates thus formed were also incubated using round bottom well plates (see FIG. 13).

3.1.5 Properties of Aggregates Formed from Stem Cells and Polymer Microspheres

In order to confirm the formation of aggregates formed of polymer microspheres attached to HAV peptides and stem cells according to the above examples, the shear modulus of each aggregate was measured using a flow meter. As a result, it was observed that the elastic modulus (G ′) of the aggregate using the polymer microspheres to which the RGD peptide and the HAV peptide were attached was higher than that of the aggregate using the polymer microspheres to which the peptide was not bound. It was found that there is a binding force between the peptide and the cell bound to the polymer, and due to this binding force it forms an aggregate of microspheres and cells (see Fig. 14). In Figure 14, "RGD-alg" represents the RGD peptide introduced polymer microspheres, "Cadherin-alg" represents the HAV peptide introduced polymer microspheres, "Alginate" represents a polymer microsphere control group without the peptide introduced, solid line is the elastic modulus ( G ') and the dotted line represent the loss modulus (G ").

3.2 Evaluation of Cell Differentiation Capacity of Aggregates Formed from Stem Cells and Polymer Microspheres

3.2.1 Evaluation of Adhesion of Stem Cells and Observation of Cell Phenotypes

Polymeric scaffolds with RGD peptides alone, which provide the binding between cells and scaffolds, to assess the binding capacity of stem cells and to identify cell phenotype and cell-cell binding using polymer microspheres that provide cell-to-cell binding. Experiment was carried out using as a comparison group.

When RGD peptides and / or HAV peptides were not incubated with polymer microspheres and stem cells, it was confirmed that relatively small amount of cells adhered and grew due to low adhesion of stem cells. In contrast, RGD peptides and / or In addition, it was observed that a large number of cells were growing in the microparticles conjugated with the HAV peptide. In the polymer microspheres in which the HAV peptides were combined alone, the cells and the cells showed the form of binding, and the cell populations could be observed. In the polymer microspheres in which the RGD peptides and the HAV peptides were simultaneously bound, the cell populations were also formed. Could be observed. In contrast, in the polymer microspheres to which RGD peptides were bound alone, it was possible to observe the cells growing on the surface of the microspheres (see FIG. 15). In Figure 15, "RGD-alginate" represents the RGD peptide introduced polymer microspheres, "CAD-alginate" represents the HAV peptide introduced polymer microspheres, "R / C-alginate" refers to the polymer microspheres to which the RGD peptide and HAV peptide is simultaneously bonded "Alginate" indicates a polymer microsphere control group without peptide.

3.2.2 Evaluation of Cell Viability of Stem Cells and Polymer Microsphere Aggregates

The survival rate of stem cells in the aggregates formed from the polymer microspheres and the stem cells was evaluated. The aggregates using the polymer microspheres to which the RGD peptide and / or the HAV peptide were not bound can be observed that the aggregates did not form because there was no binding force between the cells and the microspheres, and the cell survival rate was lower than that of the other groups. . In contrast, in the aggregates using the polymer microspheres in which the RGD peptide or the HAV peptide were respectively bound, it was confirmed that a specific volume of aggregates were formed by the binding force between the cells and the microspheres, and the cell survival rate was also high. In addition, in the aggregate using the polymer microspheres in which the RGD peptide and the HAV peptide were simultaneously bound, the aggregates were formed with high binding force, and spherical aggregates were close, and the survival rate of the cells in the aggregate was also high (see FIG. 16). In Figure 16, "RGD-alginate" represents the RGD peptide introduced polymer microspheres, "Cadherin-alginate" represents the HAV peptide introduced polymer microspheres, "R / C-alginate" refers to the polymer microspheres to which the RGD peptide and HAV peptide is simultaneously bonded "Alginate" indicates a polymer microsphere control group without peptide.

3.2.3 Evaluation of Chondrocyte Differentiation Capacity of Stem Cells and Polymer Microsphere Aggregates

To evaluate the differentiation ability of stem cells into chondrocytes using polymer microspheres in which RGD peptides that provide binding between cells and supports are introduced alone, and polymer microspheres in which HAV peptides that provide cell-to-cell binding are used. And stem cells were mixed to form aggregates and cultured under chondrocyte differentiation medium conditions for 2 weeks (DMEM / F12, 10 nM dexamethasone, 50 μg / ml L-ascorbic acid, 5 μl / ml Insulin, 10 ng / ml TGF). -beta1). As a result of histological observation after incubation for 2 weeks, it was confirmed that the aggregates were not formed in the alginate microspheres without the peptide introduced, and the HAV peptide-introduced polymer microspheres introduced with the RGD peptide alone were not used. It can be seen that the differentiation of cartilage tissues from alcian blue and sirius red, which evaluates the differentiation ability of cartilage tissues in aggregates, was more actively performed (see FIG. 17). In FIG. 17, "RGD-Alginate" represents RGD peptide-introduced polymer microspheres, "Cadherin-Alginate" represents HAV peptide-introduced polymer microspheres, and "Alginate" represents a polymer microsphere control in which no peptide is introduced.

In order to evaluate the chondrocyte differentiation ability of stem cells, gene expression levels of collagen type 2 and aggrecan, which are chondrocyte differentiation factors, were measured and compared, and as a result, RGD peptides were expressed in aggregates using high-molecular microparticles combined with HAV peptides. It was confirmed that a large amount of genes were expressed as compared with aggregates using polymer microspheres bound alone. In addition, it was confirmed that the aggregates using polymer microspheres in which RGD peptides and HAV peptides were combined at the same time have higher gene expression of chondrocyte differentiation factors than other groups. Through this, it was confirmed that stem cell culture using polymer microspheres providing cell-to-cell binding is more effective in culturing chondrocytes compared to the case of using polymer microspheres providing only cell-to-support binding (see: 18). In Figure 18, "RGD" represents the RGD peptide introduced polymer microspheres, "CAD" represents the HAV peptide introduced polymer microspheres, "R / C" represents the polymer microspheres to which the RGD peptide and HAV peptides are simultaneously bonded, "Alg" Denotes a macromolecular control group to which no peptide is introduced.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Sequence Listing First Sequence: DSCPPSPATERSYFHLFPPPPSPCTDSS

Sequence Listing Second Sequence: GGGGSHAVSS

Claims (15)

1. Polymeric cell scaffolds for forming cell colonies comprising:

   (a) a biocompatible polymeric backbone;

   (b) Cadherin adherent oligopeptide bound to the polymer backbone.
2. The cell support of claim 1, wherein the bond is an amide bond formed between the biocompatible polymer backbone and the caherin adherent oligopeptide.
3. The cell support according to claim 2, wherein the biocompatible polymer backbone comprises a carboxyl group, hydroxyl group or amino group for forming the amide bond.
4. The cell support according to claim 3, wherein the polymer backbone comprises a carboxyl group or a hydroxyl group or is surface modified to include a carboxyl group or a hydroxyl group.
5. The cell support according to claim 4, wherein the polymer backbone is at least one selected from the group consisting of an alginic acid polymer, a chitosan polymer and a hyaluronic acid polymer.
6. The cell support according to claim 1, wherein the cardherin-attached oligopeptide is an LRP5 peptide consisting of SEQ ID NO: 1 or HAV peptide consisting of SEQ ID NO: 2.
7. The cell support according to claim 6, wherein the LRP5 peptide or the HAV peptide forms a bond with a cardherin protein of the cell.
8. The cell support of claim 1, wherein the cell support further comprises (c) an integrin adherent oligopeptide bound to the polymeric backbone.
9. The method of claim 8, wherein the integrin adherent oligopeptide is RGD, PSHRN, FHRRIKA, YIGSR, KGD, RHD, NGR, SDGR, KQAGDV, LDV, DGEA, YGYYGDALR, FYFDLR, DALR, DLR, RLD, KRLDGS, IDA, IDAPS And REDV, wherein the cell support.
10. A method for culturing a cell population comprising the step of culturing the cells in contact with the cell support of any one of claims 1 to 9.
11. A composition for inducing stem cell differentiation comprising the cell support according to any one of claims 1 to 9.
12. Comprising the step of contacting the stem cell differentiation inducing composition according to claim 11 and the stem cells, and culturing in chondrocyte differentiation conditions medium, stem cell culture method for differentiation into chondrocytes.
13. A cell support according to any one of claims 1 to 9, (b) a composition for intracellular cell transplantation comprising a cell for in vivo transplantation bound to the cell support.
14. The composition of claim 13, wherein the cells for transplantation in the body are stem cells.
15. The composition of claim 13, wherein the cells for transplantation in the body are chondrocytes differentiated from stem cells.

Images