Claims
[1] A three-dimensional non-spherical micro-scaffold for cell culture and delivery prepared using a RP (rapid prototyping) system with a piezo-, bubble jet- or thermal jet-type spray nozzle, the micro-scaffold being made of a biocompatible polymer selected from the group consisting of PLA, PLLA, PGA, PGLA, PCL, chitosan, polylactides, polyglycolides, epsilon-caprolactone, polyhydroxyvaleric acid, polyhydroxybutyric acid, other polyhydroxy acids, polytrimethylene carbonate, polyamines, vinyl polymers, polyacrylic acids and their derivatives containing ester, polyethylene glycols, polydioxanones, polycarbonates, polyacetals, polyorthoesters, polyamino acids, polyphosphoesters, polyesteramides, polyfumerates, polyanhydrides, polycyanoacrylates, polyoxamers, polyurethanes, polyphosphazenes, aliphatic polyesters, poly(amino acid), copoly(ether-ester), polyakylene oxalate, polyamides, poly(iminocarbonate), polyoxaester, polyamidoesters, amine group-containing polyoxaester, polyacetal, polyalkanoate, gelatin, collagen, elastine, polysaccharide, alginate, chitin, hyaluronic acid, and combinations thereof, the micro-scaffold having a maximum diameter of 100 D or less.
[2] The micro-scaffold according to claim 1, wherein the three-dimensional structure is a circular tube, a polygonal tube, or a three-dimensional structure comprising a plurality of holes or tubular branches provided on the side of the tubes.
[3] The micro-scaffold according to claim 1, wherein the three-dimensional structure is a composite structure in which a drug transporter is connected to the th ree- dimensional structure.
[4] A micro-scaffold for cell culture or delivery prepared using a biocompatible polymer by a rapid prototyping system, the micro-scaffold having a Moebius strip-shaped, twisted band or twisted tubular structure, which has a single twist, such that one side of the strip is connected with the other side in an upside-down manner without intermittence, and thus there is no distinction between the inside and outside surfaces of the strip.
[5] A micro-scaffold for cell culture or delivery prepared using a biocompatible polymer by a rapid prototyping system, the micro-scaffold having a Moebius s trip-shaped twisted band or twisted tubular structure, which has two or more twists, such that one side of the strip is connected to the other side in an upside- down manner without intermittence, and thus there is no distinction between the inside and outside surfaces of the strip.
[6] A micro-scaffold for cell culture or transfer prepared using a biocompatible polymer by a rapid prototyping system, the micro-scaffold having a Klein bottle- or Klein jar-like twisted bottle or twisted tubular structure, in which the inlet and outlet of the structure meet each other through a surface of the outer wall of the structure, so that the inlet and the outlet are one and the same.
[7] The micro-scaffold according to any one of claims 4 to 6, which is a porous structure having one or more holes.
[8] The micro-scaffold according to any one of claims 4 to 6, which is a composite structure in which a drug transporter is connected to the micro-scaffold. |
Description
NON-SPHERICAL THREE-DIMENSIONAL MICRO-SCAFFOLD
FOR CELL CULTURE AND DELIVERY PREPARED USING
RAPID PROTOTYPING SYSTEM
Technical Field
[1] The present invention relates to a three-dimensional micro-scaffold for cell culture.
Background Art
[2] In the field of biotechnology and related technologies, various culture methods have been used to increase the number of cells. Among them, a three-dimensional cell culture technique that uses powder-type scaffolds (micro-scaffolds, micro-beads or micro-carriers) in culture media is known. In order to further improve this three- dimensional cell culture technique, the present inventors suggested a cell culture method comprising periodically repeating a static status and the moving status of the scaffold, that is, shaking, rolling, stirring, whirling, rocking, mixing, blending, rotation or the like, and a cell culture method comprising slowly increasing the amount of the scaffold, in which the cell culture methods are simultaneously or individually carried out. According to these cell culture methods, when an environment where cell proliferation is accelerated by cell-to-cell signals or interactions is properly used in cell culture and, at the same time, proliferated cells adhere to each other, it is possible to minimize the phenomenon that proliferation inhibitory signals occur while the relative ratio of an area where cells can proliferate is reduced. Also, intercellular separation for increasing cell proliferation efficiency is performed using physical force instead of using a chemical method, which is known to have a possibility of causing gene mutation (see Korean Patent Application No. 2006-0031995).
[3] In the above-described three-dimensional cell culture method, the micro-scaffold is used to provide an environment where cells can grow. The present inventors successfully constructed a micro-scaffold made of a bio-compatible polymer material, such as PLLA, PLGA or chitosan, and used the micro-scaffold in cell culture and in vivo transplantation (Korean Patent Registration No. 10-0539371).
[4] The above-described prior micro-scaffold was designed such that it can perform cell culture and in vivo injection/transplantation, and it is characterized in that it is a spherical micro-bead having a size of 10-lOOD.
[5] In the case of the above-described method in which the static status and the moving status are controlled while the amount of scaffold added is slowly increased (Korean Patent Application No. 2006-0031995), an excellent effect on culture efficiency can be obtained, however, because an increase in the amount of culture medium makes the
penetration of culture medium difficult, the scaffold and cells are precipitated in the static status, which is important in cell culture, and thus cell culture efficiency is reduced in a position spaced more than a few mm apart from the interface with the culture medium. It is believed that the reason why cell culture efficiency is reduced as described above is because the spherical shape is not suitable in the diffusion of chemical substances, the size of the scaffolds is too small for the space between the scaffolds to perform diffusion, and also the space between the scaffolds becomes narrow as the number of growing cells increases. Moreover, because the spherical internal space does not provide a surface for cell adhesion, the spherical shape is inefficient compared to a non-spherical three-dimensional structure in terms of a total area for cell adhesion. To improve such shortcomings, the present inventors attempted to construct a scaffold having fine pores therein. However, because this scaffold is not a completely porous structure, culture medium is not difficult to diffuse through the scaffold, and the rate of differentiation in the desired direction can greatly vary depending on the shape of the scaffold even in the same chemical environment (culture medium, etc). Particularly, in the case of highly hydrophilic materials, cell adhesion and proliferation rate are increased and the migration of cells to the surrounding scaffolds can easily occur. In the case of materials having low hydrophilicity, cells very rapidly proliferate to the saturation density in the phase in which cells form colonies, but the migration of cells to the surrounding scaffolds is slow. Thus, the present inventors have recognized that the shape and material of micro-scaffolds must be greatly varied depending to the kind and intended use of cells to differentiate. [6] Due to the recent development of rapid prototyping (RP) technology, nanoscale prototyping had become possible in rapid prototypes which use inorganic materials as raw materials, but prototyping devices, which use polymers, have been developed and produced for industrial purposes, and so there is little or no recognition of a need to apply the rapid prototyping technology for prototypes having a resolution of less than 1OD. Thus, rapid prototyping of micro-scaffolds (for example, a size of less than 100 D) using biocompatible polymer materials has not been substantially studied. Even in attempts to perform the rapid prototyping of scaffolds, the scaffolds are used merely in cell culture and are intended to be transplanted in vivo together with cultured cells by injection, and the scaffolds mostly have a size much larger than 10OD. Accordingly, such rapid prototypes cannot be used in three-dimensional cell culture as an efficient proliferation method and are not impossible to inject through a thin injection needle. Thus, in order for cells supported and cultured on scaffolds resulting from such rapid prototypes to be used as cell therapeutic agents for injection in clinical applications, it is required to perform additional operations, including separating cells from the scaffolds for cell culture, and if cells are not separated from the scaffolds prior to
injection, the transplantation of the cells is possible only through excision operations which involve a large amount of bleeding.
[7] Moreover, conventional polymers for rapid prototypes are not yet clinically approved or are impossible to clinically approve even in the future, and a time of about 10 years will be required until the polymers will be used in clinical applications.
[8] In order for cell therapeutic agents to be used in practical clinical applications, the need for transplantation by injection is very important, but the injection of the cell therapeutic agents is possible only by a thin injection needle having a diameter of less than 200D, because of the characteristics of human vascular vessels. Thus, if the cell therapeutic agent does not freely pass through the above-described thin injection needle, the transplantation thereof by injection becomes impossible, and a damaged b lood vessel cannot be repaired, resulting in risk. For this reason, the cell therapeutic agents should necessarily be prototypes having a diameter smaller than that of the above injection needle, and thus there is still a need to develop a micro-scaffold, which serves as an effective support for cell culture and, at the same time, can be injected in vivo.
Disclosure of Invention Technical Problem
[9] It is an object of the present invention to provide the most three-dimensional efficient micro-prototypes, which are obtained through an already developed three-dimensional micro-prototyping technology in consideration of various problems, including the diffusion of culture medium, cell adhesion efficiency, passage through injection needles, the final intended use of cells (sites to be treated: liver, heart, brain, etc.), and biocompatibility, in order for the micro-prototypes to perform all the roles of proliferation scaffold, differentiation inducers and scaffolds, and carriers for injection.
[10] Another object of the present invention is to provide suitable micro-prototypes, which can perform all the proliferation and differentiation of stem cells for biotechnology and the role of carriers for injection.
[11] Particularly, it is an object of the present invention to provide a three-dimensional structure with no distinction between the inside and outside surfaces, considering the fact that the formation of cell colonies on the same surface is achieved at a rate faster than migration to the surrounding scaffolds and that cell culture is more advantageously achieved when the inside and outside surfaces are connected with each other. Technical Solution
[12] In order to achieve the above objects, according to the present invention, there is provided a micro-scaffold for cell culture and delivery prepared by a rapid prototyping (RP) system using a piezo-, bubble jet- or thermal jet-type spray nozzle, the micro-
scaffold comprising a biocompatible polymer selected from the group consisting of PLA, PLLA, PGA, PGLA, PCL, chitosan, polylactides, polyglycolides, epsilon- caprolactone, polyhydroxyvaleric acid, polyhydroxybutyric acid, other polyhydroxy acids, polytrimethylene carbonate, polyamines, vinyl polymers, polyacrylic acids and their derivatives containing ester, polyethylene glycols, polydioxanones, polycarbonates, polyacetals, polyorthoesters, polyamino acids, polyphosphoesters, polyesteramides, polyfumerates, polyanhydrides, polycyanoacrylates, polyoxamers, polyurethanes, polyphosphazenes, aliphatic polyesters, poly(amino acid), copoly(ether-ester), polyakylene oxalate, polyamides, poly(iminocarbonate), polyoxaester, polyamidoesters, amine group-containing polyoxaester, polyacetal, polyalkanoate, gelatin, collagen, elastine, polysaccharide, alginate, chitin, hyaluronic acid, and combinations thereof, the micro-scaffold having a three dimensional structure with a maximum diameter of 100 D or less. Preferably, the micro-scaffold has the maximum diameter of 20 to 10OD.
[13] Preferably, the three-dimensional structure is a circular tube, a polygonal tube, or a three-dimensional structure, which further comprises a plurality of holes or tubular branches on the side of these tubes. Also, the micro-scaffold according to the present invention may have a composite structure in which a drug transporter is connected to the three-dimensional structure.
[14] In another aspect according to the present invention, there is provided a micro- scaffold for cell culture or delivery, which is prepared by a rapid prototyping system using a biocompatible polymer, the micro-scaffold having a Moebius strip-shaped, twisted band or twisted tubular structure, which has a single twist, such that one side of the strip is connected to the other side in an upside-down manner without in- termittence, and thus there is no distinction between the inside and outside surfaces. The micro-scaffold having this structure is the simplest structure with no distinction between the inside and outside surfaces and most efficiently employs the properties of cells, which grow only on the same surface in cell culture. This principle will be more clearly understood with reference to FIG. 13.
[15] In another embodiment, the present invention provides a micro-scaffold for cell culture or delivery prepared by a rapid prototyping system using a biocompatible polymer, the micro-scaffold having a modified Moebius strip-shaped, twisted band or twisted tubular structure, which has two twists or more, such that one side of the strip is connected to the other side in an upside-down manner without intermittence, and thus there is no distinction between the inside and outside surfaces. This structure is a modified structure, which includes the characteristic of the simplest Moebius strip and has additional twists. In this structure, one side of the strip is connected to the other side, but the strip is twisted two times or more. Thus, at any one point, the sides of the
strips cross with each other to form an 8-like shape or a ribbon shape. This structure requires a prototyping operation, which is more complicated compared to the case of the Moebius strip having a single twist, and it can be entangled. However, as the number of twists therein increases, it can be modified such that the sides thereof cross each other, and thus the distance between the scaffold surfaces spaced apart from each other is reduced, thus increasing the probability that cells migrate to other scaffolds. Accordingly, it is a scaffold shape which can provide a more advantageous effect in cell culture.
[16] In still another embodiment, the present invention provides a micro-scaffold for cell culture or delivery prepared by a rapid prototyping system using a biocompatible polymer, the micro-scaffold having a Klein bottle- or Klein jar-like twisted bottle or twisted tubular structure, in which the inlet and outlet of the structure join each other through a surface of the outer wall of the structure, and thus are one and the same. The advantages of this structure are that it can stably protect cells, ensures a sufficient cell growth space, has no distinction between the inside and outside surfaces, and thus is most efficient in terms of the properties of cells which grow along the same surface. Also, another advantage is that the diffusion of culture medium on this structure uniformly occurs without distinction between the inside and outside surfaces.
[17] Furthermore, the micro-scaffold of the present invention may be a porous structure having at least one hole.
[18] Also, the micro-scaffold according to the present invention may be a composite structure in which a drug transporter is connected to the micro-scaffold.
[19] The micro-scaffold according to the present invention can be prepared using a known rapid prototyping (RP) technique. As already known, it is currently possible to form even a nano-scale three-dimensional micro-scaffold using the three-dimensional rapid prototyping technology. In the present invention, a rapid prototyping (RP) system having three or more axes and an axial resolution of less than 5 D is used to the above- described micro-scaffold. The RP system is preferably a spray nozzle type such as a piezo, bubble jet or thermal jet in order to produce a large amount of a non-spherical three-dimensional shape having pores or holes therein using a biocompatible polymer as a raw material.
[20] As the biocompatible polymer, PLA (poly lactic acid), PLLA (poly L-lactic acid),
PGA (poly glycolic acid), PGLA (polyglycolic/lactic acid), PCL (poly e- carprolactone), chitosan and the like can be used alone or in combination. Also, other examples include polylactides, polyglycolides, epsilon-caprolactone, polyhy- droxyvaleric acid, polyhydroxybutyric acid, other polyhydroxy acids, polytrimethylene carbonate, polyamines, vinyl polymers, polyacrylic acids and their derivatives including esters, polyethylene glycols, polydioxanones, polycarbonates, polyacetals,
polyorthoesters, polyamino acids, polyphosphoesters, polyesteramides, polyfumerates, polyanhydrides, polycyanoacrylates, polyoxamers, polyurethanes, polyphosphazenes, aliphatic polyesters, poly(amino acid), copoly (ether-ester), polyakylene oxalate, polyamides, poly(iminocarbonate), polyoxaester, polyamidoesters, amine group- containing polyoxaester, polyacetal, polyalkanoate, gelatin, collagen, elastine, polysaccharide, alginate, chitin, hyaluronic acid, and copolymers or terpolymers thereof. The biocompatible polymers which can be used in the present invention as described above, including chitosan, PLA (poly lactic acid), PLLA (poly L-lactic acid), PGA (poly glycolic acid), PGLA (polyglycolic/lactic acid), PCL (poly e-carprolactone) and the like are known to be suitable for the human body through numerous clinical tests, and the use of these polymers provides an advantage of reducing the period of a clinical test, which takes about 5-10 years.
[21] The three-dimensional structure intended in the present invention may be a polyhedral structure having pores or holes for ventilation or a three-dimensional structure having various shapes of ring, band or non-spherical structure with the internal space. The micro-scafflod of such structure provides a space for cells to adhere and grow and provides a mechanical and electrical environment suitable for proliferation and differentiation of cell, thereby ensuring the optimal culture environment and efficiency. Though the shape of each scaffold can be made to be different for cells, it is basically formed to have the maximum surface area for attachment, the attachment environment with the maximum efficiency and the ventilation with the maximum efficiency so as to form the space advantageous for diffusion of the culture fluid in the scaffold or between scaffolds. Also, the scaffold is shaped to be suitable for characteristics and size of cells. Such three-dimensional structures include a circular tube, a polygonal tube, a structure having a plurality of holes or tubular branches on the side of the tube, and a polyhedron whose inner space is exposed to an external environment. Such structures are formed in combination with each other, or two or more structures are connected to each other. Also, such structures may be made of a plurality of materials having different decomposition rates.
[22] Also, when the rapid prototyping system is used, a Moebius strip-shaped or Klein bottle-shaped scaffold having at least one twist, which is used for cell culture or delivery, can be formed using a biocompatible polymer as a raw material.
[23] These three-dimensional structures are non-spherical, and thus provide a maximum surface area in which cells can adhere and grow. Also, these structures optimize a mechanical and electrical environment required for the proliferation and differentiation of cells. Moreover, these structures have a completely porous structure, which facilitates the diffusion of culture medium and the transfer of chemical signals, so that the rate of cell migration to the surrounding scaffolds is increased, thus increasing cell
culture efficiency.
[24] The above-described micro-scaffold (micro-bead) can be used in three-dimensional cell culture to perform either a cell culture process comprising periodically repeating a static status and the moving status of the micro-scaffold, or a cell culture process comprising slowly increasing the amount of the micro-scaffold, in which the cell culture processes are simultaneously or individually performed. Thus, when an environment where cell proliferation is accelerated by cell-to-cell signals or interactions is properly used in cell culture and, at the same time, proliferated cells adhere to each other, it is possible to minimize the phenomenon that proliferation inhibitory signals occur while the relative ratio of an area where cells can proliferate is reduced. Also, intercellular separation for increasing cell proliferation efficiency is performed using physical force instead of using a chemical method, which is known to have a possibility of causing gene mutation. Accordingly, the non-spherical micro-scaffold provides high culture efficiency compared to a simple spherical scaffold.
[25] Also, the above micro-scaffold consists of a three-dimensional structure having a maximum size of 20-10OD and has a ventilation space formed therein. Thus, when the micro-scaffold is used, the collection, cultured cell and the micro-scaffold and in vivo transplantation of cells can be performed in a single syringe without needing to move the cells. Specifically, cells are collected using a syringe, the piston of which can be opened and closed, and then the cells are centrifuged so as to be suitable for proliferation and are washed. The piston of the syringe is opened, and culture medium and a powder-type scaffold (micro-scaffold) are placed into the syringe, and the cells are suspended in the culture medium. In this state, the stem cells are adhered to the micro- scaffold, and the cell attached micro-scaffold is precipitated in the culture medium. In the state in which the piston is opened, the cells are cultured, and after a given time of culture, culture medium and the micro-scaffold are added to the syringe through the syringe opening. Then, the micro-scaffold is moved to allow the cells to proliferate, and the proliferation is periodically repeated. In this way, the closed loop cell culture method using the syringe can be applied. The cells, which were cultured and proliferated according to the above method, have an advantage in that they can be transplanted into the human body without causing damage to blood vessel tissue. Advantageous Effects
[26] The present invention provides a micro-scaffold for three-dimensional cell culture, which is prepared by a rapid prototyping system so as to have a ventilation space formed therein. Thus, the micro-scaffold can perform all the roles of cell proliferation scaffolds, differentiation inducers and scaffolds, and carriers for injection.
[27] The micro-scaffold structure according to the present invention has a high ability to
diffuse culture medium into the internal space thereof, includes surfaces joined with each other, and thus has high cell adhesion efficiency. Also, it has a size capable of passing through an injection needle and comprises an internal space which allows the volume of the structure to be reduced. Accordingly, it can be transplanted using an injection needle for transplantation, and thus facilitates the transplantation of target cells by injection. In addition, it is made of a material, which was found to be biocompatible through clinical tests, and thus the clinical test thereof can be reduced. Brief Description of the Drawings
[28] Further objects and advantages of the invention can be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
[29] FIGS. 1 and 2 are views showing an embodiment of the three-dimensional scaffold of a square tube type;
[30] FIGS. 3 and 4 are views showing an embodiment of the three-dimensional scaffold of a column type;
[31] FIG. 5 is a view showing an embodiment of the three-dimensional scaffold of a honeycomb type;
[32] FIG. 6 is a view showing an embodiment of the three-dimensional scaffold of a flute type;
[33] FIGS. 7 to 10 are views showing an embodiment of the three-dimensional scaffold of a Moebius band type;
[34] FIGs. 11 and 12 are views showing an embodiment of the three-dimensional scaffold of a Klein bottle type; and
[35] FIG. 13 is a view showing the difference in the efficiency of cell culture between the scaffold of the general band type and the scaffold of a Moebius band type. Best Mode for Carrying Out the Invention
[36] Now, the three-dimensional shape of the micro-scaffold is described in detail with reference to the accompanying drawings.
[37] FIG. 1 to FIG. 6 illustrate various shapes of the micro-scaffold prepared using the RP system according to the present invention.
[38] FIG. 1 is a square tube type with a hollow center, and FIG. 2 is a square tube with openings, which is prepared using a biocompatible polymer by the RP system and has a maximum size of 20 to 100D.
[39] FTG. 3 and FIG. 4 are each a cylindrical shape and FIG. 4 has openings to improve air permeability on the side wall. They are prepared using a biocompatible polymer by the RP system and formed to have a maximum size of 20 to 10OD.
[40] FIG. 5 and 6 are a honeycomb shape and a flute shape, respectively, in which spaces
formed therein ensure air permeability and provide spaces in which cells can grow. Also, the structures are similar to those of the brain, liver or nerve cells, whereby they can be effectively applied in the culture and transplantation of such cells. Each of the structures is prepared using a biocompatible polymer by the RP system and formed to have a maximum size of 20 to 10OD.
[41] FlG. 7 to FTG. 10 are tubular or band-type Moebius strip structures, in which one side of the strip is connected to other side in an upside-down manner, so that there is no distinction between the inside and outside surfaces. The structures have one twist (FlG. 7 and FlG. 8), two twists (FlG. 9) or three twists (FlG. 10). Also, the structures are each formed of a biocompatible polymer by a RP system and have a maximum size of 20 to 10OD. The structures are classified into a shape having a single twist, a shape having an even number of twists (more than two twists) and a shape having an odd number of twists (more than three twists).
[42] The Moebius strip structure is the simplest prototype with no distinction between the inside and outside surfaces and most efficiently employ the properties of cells which rapidly grow only one surface. This strip structure shows elasticity upon transplantation by injection without being entangled and can ensure the largest space with a minimum amount of scaffolds. Thus, this structure has high cell culture and proliferation efficiencies and can be transplanted in vivo by a syringe in a cultured state.
[43] FlG. 11 and FlG. 12 show scaffolds for cell culture or delivery prepared using a biocompatible polymer by the RP system, the scaffolds having the structure of the Klein bottle (or the Klein jar). These scaffolds have the shape of a twisted bottle or tube, in which the inlet and outlet of the structure meet each other through a surface of the outer wall thereof, so that the inlet and outlet are one and the same. Each of the scaffolds is prepared from a biocompatible polymer using the RP system and has a maximum size of 20-100 D.
[44] During cell proliferation, cells cannot cross the boundary between the surfaces of a scaffold and can grow only a surface of the same phase. As shown in FlG. 13, in the case of conventional band structures, cells will proliferate on only one surface of the inside and outside surfaces as a scaffold, both the surfaces cannot be used for the proliferation of cells. However, in the inventive scaffold having a shape with no distinction between the inside and outside surfaces, cells can proliferate on both the surfaces, and thus the inventive scaffold is clearly a more efficient scaffold.
[45] Accordingly, as shown in FlG. 13, the structure of the scaffold according to the present invention has a space for cell protection and growth and, at the same time has no distinction between the inside and outside surfaces. Thus, it is the most efficient scaffold for cells which proliferate along a surface. Also, the diffusion of culture medium on the scaffold is uniform and the probability that the scaffolds are entangled
with each other is low. Thus, the inventive scaffold is suitable for cells, which show relatively fast growth during the proliferation thereof and differentiate for a long period of time.
[46] The above-described three-dimensional structures, including the Moebius strip structure or the Klein bottle structure, can be prepared to have a plurality of holes therein. The plurality of such structures can be formed in combination with each other, or may be made of a plurality of materials including a drug transporter. Such structures are 3-dimentional structure, which can consider the culture and proliferation of specific cells or the physical and chemical properties of specific cells, and they will contribute to an increase in cell culture and proliferation efficiencies.