KR20130120572A - Porous 3 dimensional cell-laden scaffold and producing method thereof - Google Patents

Porous 3 dimensional cell-laden scaffold and producing method thereof Download PDF

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KR20130120572A
KR20130120572A KR1020120043567A KR20120043567A KR20130120572A KR 20130120572 A KR20130120572 A KR 20130120572A KR 1020120043567 A KR1020120043567 A KR 1020120043567A KR 20120043567 A KR20120043567 A KR 20120043567A KR 20130120572 A KR20130120572 A KR 20130120572A
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cells
porous
cell
dimensional
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김근형
안승현
이형진
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조선대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Abstract

In order to prepare a three-dimensional carrier containing cells, the present invention was carried out by modifying a conventional dispensing method to perform cross-linking between posts by spraying calcium chloride solution. To evaluate this process, osteoblast precursors were mixed in alginate solution and prepared in a three-dimensional matrix. The prepared cell-containing structures showed high porosity and uniformly designed pore size and shape. As a result of comparing the nozzle sizes, the cells were evenly distributed in the posts, and the cell viability was about 85% compared to the initial cell viability.

Description

Porous three-dimensional structure containing cells and a method of manufacturing the same {Porous 3 Dimensional Cell-Laden Scaffold and Producing Method

The present invention relates to a porous three-dimensional layered structure containing cells and a method for manufacturing the same, in order to produce a three-dimensional structure containing cells, the present inventors modified the conventional distribution method by spraying calcium chloride solution to cross-link between posts Was performed. The prepared cell-containing three-dimensional structure exhibited high porosity and uniformly designed pore size and shape. Cells were evenly distributed within the striatum and showed high cell viability compared to the initial cell viability.

In tissue engineering, research has been actively conducted to maintain, improve or restore damaged tissues and functions of the body by making and supporting cell carriers. The development of such tissue transplants and scaffolds, ie cell carriers or constructs, provides functionalized tissues in which cells can be attached and cultured three-dimensionally with advances in biotissue engineering. It is composed of a porous structure that enables attachment, differentiation and movement, and consists of biocompatible materials that can coexist with tissues in the body, and has biodegradable properties so that the support can be replaced with cells as cells grow. Should be Recently, with the development of biotissue engineering, various mechanical engineering techniques have been used to manufacture cell carriers more precisely and systematically.

Recently, cell printing techniques have been widely applied to various tissue regeneration fields because they can hold cells in desired positions, distribute cells uniformly, and efficiently transport growth factors. Fedorovich et al., Tissue Eng C-Meth. 2012 , 18, 33, B. Guillotin, F. Guillemot, Trends Biotechnol. 2011 , 29, 4183, NE Fedorovich et al., Trends Biotechnol. 2011 , 29, 601, D. Yanan et al., PNAS 2008 , 105, 9527, V. Mironov et al., Biomaterials, 2009 , 30, 2174]. For successful tissue regeneration, the cell-loaded carrier must have high porosity, pore size control, and 100% interconnection between the pores for oxygen and nutrient supply and angiogenesis [A. Ovsianikov et al., Biomacromolecules 2011 , 12, 858, R. Gaetani et al., Biomaterials 2012 , 33, 1790]. In particular, the pore size, shape, and interconnectivity that influence new tissue formation and angiogenesis are important parameters for bone tissue regeneration [SJ Hollister, Nature Materials 2005 , 4, 518].

Several methods have been introduced to produce cell-carried carriers, such as inkjet-based printing, direct freeform fabrication, and laser-induced forward transfer [NE]. Fedorovich et al., Tissue Eng C-Meth. 2012 , 18, 33, A. Ovsianikov et al., Biomacromolecules 2011 , 12, 858, R. Gaetani et al., Biomaterials 2012 , 33, 1790, KB Fonseca et al., Acta Biomater . 2011 , 7, 1682, DL Cohen et al., Tissue Eng C-Meth . 2011 , 17, 239]. Recently, a porous structure carrier designed using a laser assisted bioprinting method has been prepared, in which fibroblasts and keratinocytes are printed using collagen solution [L. Koch et al., Biotechnol. Bioeng. 2012 , DOI 10.1002 / bit. 24455]. Gaetani et al. Incorporated human cardiac-derived cardiomyocyte progenitor cells (hCMPCs) into alginate carriers to obtain constructs with cardiogenic potential for in vitro or in vivo applications. . These cells were well distributed in the carrier and showed high cell viability (up to 92%) [R. Gaetani et al., Biomaterials 2012 , 33, 1790].

Although the survival rate of the cell-embedded carrier is being improved by newly developed techniques, the carrier fabrication technique overcomes problems such as low cell density, uneven cell distribution, weak mechanical properties, and insufficient three-dimensional pore shape. More research is needed [B. Guillotin, F. Guillemot, Trends Biotechnol. 2011 , 29, 4183, RK Pirlo et al., Biotechnol. Bioeng . 2012 , 109, 273]. In particular, as pointed out by Pirlo et al., One of the main problems is the low structure-forming ability of laminated hydrogels, which can limit the three-dimensional structure size and cause deformation of the printed carrier [RK Pirlo et al., Biotechnol. Bioeng . 2012 , 109, 273].

Hydrogels, including polyethyleneglycol, alginate, collagen and gelatin, have been widely used in the preparation of cell-embedded carriers because of their high water content, excellent biocompatibility, control of mechanical properties, and excellent new degradability [AR Costa Pinto et al., Biomacromolecules 2009 , 10, 2073. For this reason, hydrogels are well suited for the manufacture of cell-loaded structures and can be printed directly to obtain various types of tissue regeneration backbones.

Alginate, a type of typical hydrogel, is a biocompatible polysaccharide that has been extensively studied to cover various growth factors and cells since it solidifies rapidly in calcium chloride solution. Due to its ability to promote epithelialisation and granular tissue formation, alginate has been widely used in a variety of carriers for tissue regeneration such as skin, bone and blood vessels [X. Li et al., Biotechnol. Prog. 2006 , 22, 1683, T. Hashimoto et al., Biomaterials 2004 , 25, 1407. E. Alsberg et al., J. Dent. Res . 2001 , 80, 2025.]. Laminated drops easily control the pore structure (pore size and shape, porosity and permeability), so some carriers have used this manufacturing strategy. Alginates are widely used in molding and printing techniques to produce solid implants of precisely shaped cells seeded by SC Chang et al., J Biomed Mater Res . 2001, 55, 503, JJ Ballyns et al., Tissue Eng Part A. 2008, 14, 1195]. Using microfabrication techniques to produce alginate carriers seeded with cells and embedded in pores [DL Cohen et al., Tissue Eng . 2006, 12, 1325] and it is also possible to use these voids to guide tissue internal growth in vivo [NW Choi et al., Nature Mater . 2007, 6, 908, Y. Zheng et al., Biomaterials, 2011, 32, 5391]. However, it is currently not possible to directly print alginate carriers or microstructures seeded with macro-sized cells containing pores. In addition, the cells in the carrier in the three-dimensional bulk structure will die due to the low permeability of nutrients and oxygen. For this reason, there is a need to study three-dimensional cell-containing carriers capable of controlling pore size in order to study cell behavior, tissue function and regeneration [L. Koch et al., Biotechnol. Bioeng. 2012 , DOI 10.1002 / bit. 24455].

Existing three-dimensional cell structure is used by dividing the process of constructing the cell, attaching and culturing the structure, it is difficult to grow the cells evenly due to the non-uniform distribution of cells in the structure, and practical three-dimensional pores due to the hydrophilic properties of the structure-producing material There was a difficulty in producing the structural form. It is an object of the present invention to provide a structure of a three-dimensional pore structure in which cells are uniformly distributed and a method of manufacturing the same.

In the conventional rapid prototyping method, since it is difficult to process a low viscosity biocompatible solution into a three-dimensional form, in order to overcome the limitations of the method for preparing a biomedical cell structure, the present inventors generate fine water droplets containing a crosslinking solution. By spraying it on the low-viscosity polymer solution strut that is being manufactured in three-dimensional form, the surface of the strut is cured. Invented was a method of manufacturing a three-dimensional layered structure containing cells.

In the present invention, by combining a rapid prototyping (RP) process and a spraying system, the crosslinking solution is sprayed with fine water droplets on the outside of the cell carrier to cure the support surface during the preparation of the cell carrier using low viscosity natural polymer. Cross-linking solved the problems presented in the conventional method and produced a cell carrier in the form of 3D pores in which cells are uniformly contained. The prepared cell carrier can be cultured according to the characteristics of the cell carrier without undergoing cell seeding and the like, and the cell carrier can be manufactured according to the patient's situation during the surgical procedure by using autologous cells. It is considered to be a method capable of producing a cell carrier.

The present inventors prepared alginate carriers loaded with osteoblastic precursor MC3T3-E1, which were designed to have high porosity and uniform pore size and shape. The new carrier of the present invention has been produced in a new way including the spraying process by modifying the conventional biocompatible material dispensing method. Two different size distribution nozzles (310 μm and 610 μm) were used to measure the distribution of cells in the strut and cell viability in the carrier. This three-dimensional cell distribution technology of the present invention was able to successfully prepare a new three-dimensional alginate carrier.

The calcium chloride solution was sprayed on the alginate solution dispensed to produce the stacked cell-containing alginate struts using a spraying member to crosslink. The calcium chloride (2% by weight) solution was mixed with PBS and sprayed using a spraying member to cross-link the cell-containing alginate struts. As a result, the sprayed calcium chloride solution was sprayed onto the alginate struts [FIG. 1 b]. If the calcium chloride concentration was less than 2% by weight, the crosslinking of the alginate struts was very low, and the structure thus produced was very unstable. This simple process of the present invention significantly improves the three-dimensional contour of the alginate struts by allowing the outer shells of the alginate struts to crosslink with each other.

The image of Figure 2 illustrates this process. 2 a and d show the initial design of the two-layer column and four-layer strut, the pore size being 500 μm and the strut size being 310 μm. 2 b and c show a two-layered alginate carrier, respectively (b) prepared by using a conventional distribution method and (c) prepared by spraying the calcium chloride solution without spraying the calcium chloride solution. In addition, e and f of FIG. 2 show the four-layered alginate support | carriers which were prepared (f) by adding (e) and a spraying process, respectively, without a spraying process. As shown in the photo, spraying the calcium chloride solution forms a three-dimensional structure and the pores are arranged in the alginate carrier, which retains its shape better than the carrier prepared by a conventional dispensing process. 2 g is a graph showing the injection efficiency, the head office efficiency is defined as the ratio of the total area of the prepared pores to the total area of the pores originally designed in the four-layer carrier. As shown in the figure, the pore size of the carrier was stabilized when the calcium chloride was 2% by weight or more. Based on this data, we prepared the carrier by spraying a 2% calcium chloride solution. Figure 2 h and i are photographs of the four-layer alginate carrier prepared (h) with or without spraying the calcium chloride solution (i).

The pore arrangement (pore size and porosity) of the carrier has a significant effect on cell activity because it provides cell migration, nutrients, and pathways of waste products [FJ O'Brien et al., Biomaterials 2005 , 26, 441, CM Murphy et al., Biomaterials 2010 , 31, 466]. 3 is a micrograph of the pore structure of a 20 × 20 × 4.5 mm 3 size alginate carrier loaded with cells (MC3T3-E1). Although the thickness of the alginate carrier embedded with cells exceeded 4.5 mm, the carrier morphology was well formed and the designed pores were well maintained [FIG. 3D]. The three-dimensional pore structure of the alginate carrier had a passageway extending unobstructed from the top to the bottom, as shown in the internal photograph of FIG. Although the pores originally designed were perfectly rectangular, the result was a slightly rounded shape due to the dissolution of the alginate struts at the contact of each strut.

Printed samples were stained with calcein AM and ethidium homodimer 1 to determine cell viability in alginate carriers, respectively. 3 (e) and 3 (f) are fluorescence photographs of the carrier, with viable cells green and dead cells red. As shown, the cells were well distributed throughout the three-dimensional alginate carrier.

Two different nozzle sizes (outer diameter 310 μm and 610 μm) were compared to determine the effect of nozzle size on cell viability. Cell viability was measured during this process by simultaneously dispensing cells in a 3.5 wt% alginate solution. The alginate solution was carefully mixed with the cells (3.15 × 10 6 ml −1 ) and then the mixed solution was placed on the surface of the distribution member. 4 (a) and 4 (b) show alginate carriers containing cells made from two nozzles of different sizes, respectively. The strut sizes (diameter) made with 310 μm and 610 μm nozzles were 430 ± 36 μm and 660 ± 43 μm, respectively. In the figure, in the fluorescence images of two carriers having different pore sizes, green represents living cells and red represents dead cells. As shown in the photograph, the cells were uniformly distributed throughout the alginate strut. Cell viability in the dispensing process was 85 ± 3% [610 μm, FIG. 4 (a)] for large nozzles and 84 ± 5% [310 μm, FIG. 4 (b)] for small nozzles. The cell viability was high, although cells were ejected using high pneumatic pressure (40 and 210 kPa) in the dispensing vessel. In addition, even if the nozzle sizes were different, there was almost no difference in survival rate.

Confocal microscopy to determine the spatial distribution of cells (FIGs. 4 (c) and (d) for nozzle sizes 310 μm and 610 μm, respectively) shows that cells on both carriers are uniformly well inside the alginate struts. It was distributed. Fluorescence microscopy and confocal microscopy data showed that osteoblast precursors were uniformly distributed in the alginate carrier, and cell viability was suitable for tissue regeneration. In addition, although the alginate carrier containing the primary cultured chondrocytes was not photographed, the survival rate of these cells was higher than that of the osteoblast precursors, which resulted in 91% value when produced with a 310 μm nozzle.

Since this process of the present invention provides a safe environment for the cells contained in the carrier, the prepared cell-containing carrier has produced a three-dimensional pore structure composed of reticular posts loaded with cells.

The present invention

a) mixing the cells in a biocompatible material solution;

b) dispersing the biocompatible material solution in which the cells are mixed in a rapid molding method to form a strut layer on the stage and simultaneously forming a crosslink in the biocompatible material to be distributed to prepare a crosslinked strut layer. Doing;

c) distributing the biocompatible material solution in which the cells are mixed by the rapid prototyping method on the crosslinked support layer, and simultaneously forming a crosslink in the biocompatible material to deposit the crosslinked support layer; And

d) repeating step c); provides a method for producing a porous three-dimensional laminated carrier containing a cell comprising a.

In addition, the present invention is the biocompatible material is fucoidan, collagen, alginate, chitosan, hyaluronic acid, silk, polyimides, polyamix acid, polycarprolactone, polyetherimide , Nylon, polyaramid, polyvinyl alcohol, polyvinylpyrrolidone, polybenzyl-glutamate, polyphenylene terephthalamide, polyaniline (polyaniline), polyacrylonitrile, polyethylene oxide, polystyrene, cellulose, polyacrylate, polymethylmethacrylate, polylactic acid; PLA), polyglycolic acid (PGA), copolymer of polylactic acid and polyglycolic acid (PLGA), poly {poly (ethyleneoxa D) terephthalate-co-butylene terephthalate} (PEOT / PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester { poly (ortho ester; POE}, poly (propylene fumarate) -diacrylate; poly (propylene fumarate) -diacrylate; PPF-DA} and polyethylene glycol diacrylate; PEG-DA} It provides a method for producing a porous three-dimensional laminated carrier containing cells characterized in that at least one selected from the group consisting of.

In addition, the present invention is a porous three-dimensional stack containing cells, characterized in that the cross-linking of the steps b) and c) is carried out using at least one of a crosslinking solution spraying method and a photocrosslinking method of irradiating UV Provided is a method for preparing a carrier.

In addition, the present invention the crosslinking solution is gypsum; Or apatite hydroxide, apatite carbonate, fluoride apatite, chlorine apatite, α-TCP, β-TCP, calcium metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium pyrophosphate, calcium carbonate, calcium sulfate, EDC { 1-ethyl- (3-3-dimethylaminopropyl) carbodiimide hydrochloride} or a mixture of one or more mixtures thereof.

The present invention also provides a method for spraying the crosslinking solution by applying ultrasonic waves to the crosslinking solution, a method for injecting a crosslinking solution using an electrosprayer, a method for aerosolizing and injecting a crosslinking solution by applying air pressure, and crosslinking. It is characterized in that it is carried out by one or more methods selected from aerosolizing and spraying by applying heat to the solution.

In the description and claims of the present invention, "injection" means spraying the solution into particles having a fine particle diameter, such as fog. More specifically, in the specification of the present invention, "injection" refers to applying a droplet made by applying heat or ultrasonic waves to the surface of a strut, such as fine water particles generated in a humidifier, and adding an aerosol container with a high pressure gas. The fine crosslinking solution particles are sprayed when they are injected into the support surface, and the fine crosslinking solution particles are sprayed onto the support surface, and droplets are sprayed with a nebulizer or an electrosprayer. It is used in a broad sense, including various injection methods known to the person.

The present invention also provides a porous three-dimensional laminated carrier or structure containing cells, prepared by the above method, 100% pore interconnectivity.

In addition, the present invention is characterized in that the cell viability of the carrier is 70% or more.

The present invention also provides a therapeutic agent for cell or tissue regeneration comprising a porous three-dimensional layered structure containing the cells. The therapeutic agent for regenerating cells or tissues including the porous three-dimensional layered structure containing the cells of the present invention can be used for the treatment of various types of tissues and cells, including bone tissue, epithelial tissue, connective tissue, and neural tissue, including cartilage tissue. Some examples include porous three-dimensional laminated structures in which the cells of the present invention are included in all diseases that require the treatment of diseases caused by bone damage or cartilage tissue damage, baldness treatment, cosmetic surgery, general plastic surgery and tissue regeneration. The therapeutic agent for cell regeneration including the same can be applied. In addition, in the present invention, the term "cell" refers to a cell used for regeneration of a cell or tissue, and in addition to various adult cells including skin cells, blood vessel cells, osteocytes, and chondrocytes, various cells such as stem cells and monocytes. It was used in a broad sense, including cells that can differentiate into cells.

The present invention provides a therapeutic agent for bone tissue regeneration comprising a porous three-dimensional laminate carrier containing at least one of bone cells and chondrocytes.

The present invention provides a therapeutic agent for skin cell regeneration comprising a porous three-dimensional layered carrier containing skin cells.

The present invention also provides a therapeutic agent for cell regeneration comprising a porous three-dimensional layered carrier containing vascular cells.

The cell-containing porous three-dimensional cell carrier prepared by the method of the present invention had a stable three-dimensional structure, large pore size, and 100% interconnection between pores, thus supplying oxygen and nutrients necessary for cell survival and growth. This smoothly made cell viability was very good at least 70% or more.

1 is a schematic diagram of the method of the present invention. (a) shows the concept of complementing the cell loading process with the spraying process using CaCl 2 solution. (b) shows the process of crosslinking with CaCl 2 solution sprayed on alginate struts.
Figure 2 (a) is a two-layer structure designed, (b) is an alginate structure not sprayed CaCl 2 solution, (c) is an alginate structure sprayed CaCl 2 (2% by weight) solution. (d) is a schematic diagram showing the fourth floor, (e) is a non-alginate structure spraying CaCl 2 solution, (f) is an alginate structure spraying CaCl 2 (2% by weight) was added. (g) is a graph showing the spraying efficiency according to various weight ratios of CaCl 2 solution. (h) is the final structure of the four-layer alginate without spraying CaCl 2 solution, and (i) is the alginate structure with spraying CaCl 2 (2 wt.%) solution.
3 is a three-dimensional shape (20 × 20 × 4.5 mm 3) of an alginate carrier of uniform pore size (435 ± 32 μm) and uniform strut size (355 ± 25 μm). (a) is a top view from above; (b, c) is a side photograph; (d) is an enlarged photograph of the surface and cross section of (b); 9e) are live cells (green) and (f) dead cells (red).
4 is a panel of survival (green) and death (red) of the same portion in the survival test. (a) is a cell sized alginate carrier with a nozzle size of 610 μm and (b) with a nozzle size of 310 μm. Confocal microscopy images of viable and dead cells of a carrier made with two different nozzles {(c) 610 μm, (d) 310 μm}.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is obvious to those skilled in the art that the scope of the present invention is not limited only to the description of the embodiments.

cell

MC3T3-E1 cells were provided by Professor Claudia Fischbach-Teschl (Cornell University, Ithaca, NY, USA). To maintain the cells, MC3T3-E1 mouse cranial osteoblasts were cultured and maintained in α-minimal essential medium (Life Science, USA) containing 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA). .

Preparation of Alginate Solution Containing Cells

Alginate hydrogels for cell distribution were prepared according to the prior art (CS Lee, JP Gleghorn, NW Choi, M. Cabodi, AD Stroock, LJ Bonassar, Biomaterials 2007 , 28, 2987.). Low viscosity, high G-content non-medical grade LF10 / 60 alginate (FMC BioPolymer, Drammen, Norway) was mixed with PBS to prepare 3.5 wt% alginate. Before loading the cells, 0.5 wt% CaCl 2 (Sigma-Aldrich, St. Louis, Mo., USA) was added to the alginate solution to increase the viscosity of the solution, and the mixing ratio of the alginate and CaCl 2 solution was 7: 3. The cells were mixed with the alginate solution at a density of 3.15 × 10 6 ml −1 using a three-way stopcock tool. The cell / alginate mixture was placed in a syringe container. A secondary crosslinking process was performed with 2% CaCl 2 solution (in PBS) to ensure complete crosslinking of the cells embedded in the alginate carrier.

Preparation of Alginate Carrier Containing Cells

A computer-controlled three-axis robot system (DTR2-2210T, Dongbu Robot, Bucheon, South Korea), with the addition of a dispensing member and a spraying member (Tess-7400; Paju, South Korea), was used to provide a multi-layered porous alginate carrier containing cells. Prepared. 2 wt% CaCl 2 solution was continuously sprayed on the carrier by the ultrasonic spray member during the manufacturing process (injection rate: 14.5 ± 2.2 ml / min). Starting from the bottom, each layer was arranged at right angles to the layer immediately below to form a 0 ° / 90 ° support structure. A three-dimensional square carrier was layered using a robotic system that ejects alginate containing cells as a support on the plate.

The pore size and strut size are important factors in the multilayer structure because it can affect not only the physical properties but also the cellular activity of the finally prepared carrier. In order to produce a cell-containing carrier, the inventors designed the ejection of alginate through two nozzles of different diameters (outer diameter = 310 µm and 610 µm, inner diameter = 150 µm and 330 µm) in a distribution system, As a result, different sized posts were prepared. In order to observe the effect of the nozzle size, the distance between the struts and the distance between the holes was fixed at 620 μm. In order to obtain a homogeneous alginate strut shape, the inventors fixed the dispensing conditions to temperature = 26.5 ° C., ejection pressure of large nozzles = 40 kPa, ejection pressure of small nozzles = 210 kPa, nozzle movement speed = 10 mm / s. The multi-layered porous alginate structure containing the cells was plotted and then immersed in 2 wt% CaCl 2 solution to fully cure the carrier. The carrier was then washed twice with PBS solution. Table 1 shows the conditions of this process. The alginate carrier had to be recrosslinked with CaCl 2 to obtain an alginate carrier loaded with cells. Secondary crosslinking processes were performed with various concentrations of CaCl 2 solution (1 wt.%, 2 wt.% And 3 wt.%). The ejected alginate carrier was immersed in the solution for 1 minute. In order to obtain appropriate secondary crosslinking conditions, the inventors observed changes in the three-dimensional shape of the carrier at different crosslinking times (1 day and 14 days). After 14 days of immersion, the structural state of the alginate carrier was well maintained when the CaCl 2 concentration was 2% by weight or more.

Figure pat00001

Cell viability measurement

Carriers containing the cells were prepared and then exposed to 0.15 mM calcein AM and 2 mM ethidium homodimer-1 for 45 minutes in an incubator. Stained carriers were analyzed with a fluorescence microscope (TE2000-S; Nikon, Tokyo, Japan) equipped with a SPOT RT digital camera (SPOT Imaging Solutions, Sterling Heights, MI, USA). Images were captured with Image J program (NIH, Bethesda, MD, USA) to count cell viability and green and red spots were counted. The survival rate of the alginate carrier containing the cells was then determined. The ratio of viable cell number to total cell number was calculated using software and the ratio was normalized to the initial cell viability before cell-alginate ejection. Initial survival was determined using Trypan Blue (Mediatech, Herndon, VA, USA). Confocal microscopy (LSM700; Carl Zeiss, Wetzlar, Germany) was used to obtain stereoscopic images of viable cells (green) and dead cells (red) in alginate struts.

Claims (11)

a) mixing the cells in a biocompatible material solution;
b) dispersing the biocompatible material solution in which the cells are mixed in a rapid molding method to form a strut layer on the stage and simultaneously forming a crosslink in the biocompatible material to be distributed to prepare a crosslinked strut layer. Making;
c) distributing the biocompatible material solution in which the cells are mixed by the rapid prototyping method on the crosslinked support layer, and simultaneously forming a crosslink in the biocompatible material to deposit the crosslinked support layer; And
d) repeating step c); comprising a cell comprising a porous three-dimensional laminated carrier manufacturing method.
The method of claim 1,
The biocompatible materials are fucoidan, collagen, alginate, chitosan, hyaluronic acid, silk, polyimides, polyamix acid, polycarprolactone, polyetherimide, nylon , Polyaramid, polyvinyl alcohol, polyvinylpyrrolidone, polybenzyl-glutamate, polyphenylene terephthalamide, polyaniline, polyaniline Acrylonitrile, polyethylene oxide, polystyrene, cellulose, polyacrylate, polymethylmethacrylate, polylactic acid (PLA), Polyglycolic acid (PGA), copolymers of polylactic acid and polyglycolic acid (PLGA), poly {poly (ethylene oxide) terephthalate Tri-co-butylene terephthalate} (PEOT / PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester {poly (ortho group consisting of ester; POE}, poly (propylene fumarate) -diacrylate; PPF-DA} and polyethylene glycol diacrylate; PEG-DA Method for producing a porous three-dimensional laminate carrier containing cells characterized in that at least one selected from.
The method of claim 1,
Forming the crosslinking of step b) and step c) is a method for producing a porous three-dimensional laminated carrier containing cells, characterized in that performed using at least one of a crosslinking solution injection method and a UV light irradiation method.
The method of claim 3,
The crosslinking solution is gypsum; Or apatite hydroxide, apatite carbonate, fluoride apatite, chlorine apatite, α-TCP, β-TCP, calcium metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium pyrophosphate, calcium carbonate, calcium sulfate, EDC { 1-ethyl- (3-3-dimethylaminopropyl) carbodiimide hydrochloride} or a method for producing a porous three-dimensional laminated carrier containing cells, characterized in that a solution of at least one mixture selected from their salts.
The method of claim 3,
The crosslinking solution injection may be performed by applying ultrasonic waves to a crosslinking solution, a method of spraying a crosslinking solution using an electrospray, a method of aerosolizing by applying air pressure to a crosslinking solution, and applying heat to the crosslinking solution Method for producing a porous three-dimensional laminated carrier containing cells, characterized in that carried out by one or more methods selected from aerosolization method of spraying.
A porous three-dimensional laminated carrier comprising a cell prepared by the method of any one of claims 1 to 5, wherein the pore interconnectivity is 100%.
The method according to claim 6,
The carrier is characterized in that the cell viability is 70% or more, porous three-dimensional laminated carrier containing cells.
A therapeutic agent for regenerating cells or tissues comprising a porous three-dimensional layered structure containing the cells of claim 6.
9. The method of claim 8,
A therapeutic agent for regeneration of osteocytes or chondrocytes comprising a porous three-dimensional laminated carrier containing at least one of osteocytes and chondrocytes.
9. The method of claim 8,
A therapeutic agent for skin cell regeneration comprising a porous three-dimensional layered carrier containing skin cells.
9. The method of claim 8,
A therapeutic agent for cell regeneration comprising a porous three-dimensional layered carrier containing vascular cells.
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