WO2013027962A2 - Scaffolded assembly for in vitro expansion of haematopoietic stem cells or precursor cells, and perfusion bioreactor and bioreaction system using same - Google Patents
Scaffolded assembly for in vitro expansion of haematopoietic stem cells or precursor cells, and perfusion bioreactor and bioreaction system using same Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- A—HUMAN NECESSITIES
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- 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
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A—HUMAN NECESSITIES
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- 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
- A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2533/30—Synthetic polymers
- C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
Definitions
- the present invention relates to a scaffold assembly for in vitro expansion of hematopoietic stem or progenitor cells, a perfusion bioreactor and a bioreaction system for in vitro expansion of hematopoietic stem or progenitor cells using the scaffold assembly.
- Hematopoietic stem cells refer to cells having the ability to differentiate into blood cells such as red blood cells, white blood cells, and platelets, which constitute blood. Hematopoietic stem cells are produced in large quantities especially in the bone marrow and present in about 1% of normal bone marrow cells. It has self-renewing capacity, high capacity for cell division, and multi-potential differentiation capacity that are characteristic of stem cells (van der Kooy D, etc.). 2000, science, 287; 1439-1441).
- hematopoietic stem cells are insufficient, such as aplastic anemia, if hematopoietic stem cells are sick, such as leukemia, myelodysplastic syndrome, etc.
- the parent cell cannot function properly and cannot produce blood cells.
- hematopoietic stem cell transplantation if the hematopoietic stem cells are completely removed and the normal hematopoietic stem cells are administered to the patient, the hematopoietic stem cells migrate to the patient's bone marrow and regenerate normal blood cells to restore normal hematopoietic function. It is called hematopoietic stem cell transplantation.
- Source materials that can provide hematopoietic stem cells include bone marrow, umbilical cord blood, and peripheral blood. When hematopoietic stem cell transplantation is performed using each of them, each is referred to as bone marrow transplantation, cord blood transplantation, and peripheral blood transplantation. The type will depend on the condition of the donor and the availability of the donor.
- hematopoietic stem cells are also very small cells in the bone marrow in the human body. Therefore, the development of a technique for obtaining a large amount of hematopoietic stem cells that we need for hematopoietic stem cell transplantation is very useful.
- cord blood-derived hematopoietic stem cells and hematopoietic progenitor cells have been reported to be capable of sufficient in vitro growth when co-cultured with a cell feeder layer in the presence of recombinant cytokines.
- Slovick FT et al. (1984) Exp Hematol 12: 327-338
- Kohler T et al. (1999) Stem Cells 17: 19-24
- McNiece IK et al. (2002) Exp Hematol 30: 612-616.
- Recombinant cytokines used for this purpose include Flk-2 / Flt-3 ligand (FL), stem cell factor (SCF), thrombopoietin (TPO), granulocyte-colony stimulating factor (G-CSF), megakaryocyte growth and development factor (MGDF), interleukin-3 (IL-3), and interleukin-6 (IL-6) [Petzer AL, et al. (1996) J Exp Med 183: 2551-2558; McNiece I, et al. (2000) Exp Hematol 28: 1181-1186; Conneally E, et al. (1997) Proc Natl Acad Sci U S A 94: 9836-9841; Ueda T, et al. (2000) J Clin Invest 105: 1013-1021.
- FL Flk-2 / Flt-3 ligand
- SCF stem cell factor
- TPO thrombopoietin
- G-CSF granulocyte-colony stimulating factor
- hematopoietic stem cells are lost through apoptosis when cultured in vitro.
- hematopoietic stem cells were frozen and thawed again for storage of the collected hematopoietic stem cells, more hematopoietic stem cells would undergo cell death. Due to this, the conventional method has a limit of proliferating hematopoietic stem cells only about 30 to 40 times in 7 days.
- An object of the present invention is to provide a scaffold assembly for in vitro growth of hematopoietic stem cells or progenitor cells of a structure similar to osteoblastic niche.
- the present invention also aims to provide a perfusion bioreactor and bioreaction system for in vitro growth of hematopoietic stem or progenitor cells using the scaffold assembly to provide an environment similar to the environment in the body.
- the scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells of the present invention comprises one or a plurality of nanofiber sheets; And one or a plurality of lattice scaffolds stacked between the nanofiber sheets.
- the nanofiber sheet is characterized in that it is produced by electrospinning.
- the nanofiber sheet is made of poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid). ) (PGA), poly (caprolactone) (PCL), poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, derivatives and copolymers thereof It is characterized by consisting of a suitable or biodegradable polymer.
- the lattice structure scaffold is characterized by being produced by rapid prototyping.
- the lattice scaffolds are poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly ( Glycolic acid) (PGA), poly (caprolactone) (PCL), poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethylenecarbonate, derivatives and copolymers thereof
- biodegradable ceramics selected from biocompatible or biodegradable polymers and bioactive glass, hydroxyapatite, tricalcium phosphate (TCP) and porcine bone powder.
- the present invention also comprises a scaffold assembly for in vitro growth of the hematopoietic stem or progenitor cells of the present invention; Cells anchored to a scaffold of the scaffold assembly; And a perfused bioreactor comprising a liquid contained for carrying oxygen.
- the perfusion bioreactor of the present invention is characterized in that it comprises an oxygen carrier liquid inlet, an oxygen carrier liquid outlet and a receptacle for receiving the scaffold assembly.
- the scaffold assembly is characterized in that the receiving portion for receiving the scaffold assembly is accommodated in the scaffold assembly receiving portion in a sliding manner without a separate adhesive or fixing agent.
- the cells are characterized in that hematopoietic stem cells or progenitor cells.
- the present invention also provides a perfusion bioreactor according to the present invention;
- a pump connected with the perfusion bioreactor to create a flow with constant vibration in the perfusion bioreactor;
- a culture medium supply unit for supplying a culture medium to the perfusion type bioreactor;
- it provides a biological reaction system for in vitro growth of hematopoietic stem cells or progenitor cells comprising a control unit for controlling the culture medium supply rate, the number of vibrations applied into the perfusion type bioreactor.
- the bioreaction system for in vitro growth of hematopoietic stem or progenitor cells of the present invention comprises a biomass removal device for removing biomass from an oxygen carrier liquid from the perfusion bioreactor, a synthetic or semi-synthetic component of an oxygen carrier liquid. From a group consisting of a separation device for separating carbon dioxide from other components, a carbon dioxide removal device for removing carbon dioxide from an oxygen carrier liquid, an oxygen addition device for adding oxygen to the oxygen carrier liquid, and an air removal device for deaeration of the oxygen carrier liquid. It further comprises one or more selected devices.
- the scaffold assembly of the present invention and the perfusion reactor using the same are made of materials having biocompatibility, chemical compatibility and mechanical compatibility, and are three-dimensional for cell growth by rapid prototyping. Composed of nanosheets and a scaffold that provides an environmental environment, it is structurally similar to an osteoblastic niche, and a certain size of percussion is pumped by the pump without losing the cells fixed to the scaffold. It is possible that the cells immobilized on the scaffold are exposed to a constant stimulus to provide an optimal environment similar to that in vivo for in vitro growth of hematopoietic stem or progenitor cells.
- FIG. 1 shows an osteoblastic niche scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells in which one nanofiber sheet and one lattice scaffold are stacked according to the present invention.
- Figure 2 shows a perspective view and a side view of a scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells stacked with a plurality of nanofiber sheets and a plurality of lattice scaffold according to the present invention.
- Figure 3 shows a perfusion reactor for in vitro growth of hematopoietic stem or progenitor cells according to the present invention.
- Figure 4 shows the state in which the scaffold assembly is accommodated in the scaffold receiving portion of the perfusion reactor for in vitro growth of hematopoietic stem or progenitor cells according to the present invention.
- FIG. 5 shows a biological response system for in vitro growth of hematopoietic stem or progenitor cells according to the present invention.
- FIG. 1 illustrates a scaffold assembly 100 for in vitro growth of hematopoietic stem or progenitor cells according to the present invention.
- the scaffold assembly 100 is formed by sequentially stacking one nanofiber sheet 20 and one lattice scaffold 10.
- one nanofiber sheet 20 and one lattice-type scaffold 10 are sequentially stacked to grow hematopoietic stem cells. It forms an osteoblastic niche structure similar to the environment in the body.
- Hematopoietic stem cell niche refers to a specialized microenvironment made by supportive cells expressing cell membrane-binding proteins or secretory factors that regulate the mobility, dormancy and differentiation of hematopoietic stem cells, and promote maintenance and autologous hematopoietic stem cells.
- the concept of niches was introduced by schofield in 1978.
- Hematopoietic stem cell niches are typically known as osteoblastic niche and vascular niche, and the lattice scaffold 10 of the present invention serves as the osteoblastic niche and is a hematopoietic Provide an environment suitable for blast growth.
- the lattice-type scaffold 10 is formed by a rapid molding method to create a micro environment (niche) favorable for the growth of hematopoietic stem cells to immobilize the cells for bone tissue regeneration Serve as a support
- the nanofiber sheet 20 is laminated to the lattice scaffold and then accommodated in the scaffold assembly receiving portion, and is periodically circulated by a perfusion reactor with a pump.
- the percussive stimulation of percussion by a continuous pump not only acts as a barrier to prevent the cells from being swept by the percussion while the cell is incubated, but also shows the advantage of easy loading of growth factors for hematopoietic stem cell growth due to its large surface area. .
- the scaffold assembly of the present invention has both nano size pores and micro size pores to provide an optimal environment for hematopoietic stem cell growth. That is, the nanofiber sheet is formed in the form of three-dimensional interconnected nano-size pores in the range of 1 to 100 nm, the lattice scaffold is formed in the form of three-dimensional interconnected pores in the range of 100 to 1000 ⁇ m
- the pores of various sizes are included in the scaffold assembly of the present invention to be similar to the living environment, and the percussion stimulus by the pump is evenly delivered while supplying the affected substances and oxygen to the individual cells efficiently, thereby preventing efficient cell proliferation and differentiation and cell necrosis. To effect such as.
- the lattice scaffold 10 includes poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL), At least one or more biocompatible or biodegradable polymers selected from the group consisting of poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethyl carbonate, derivatives and copolymers thereof, It is prepared to have a three-dimensional pore structure using a material comprising a biodegradable ceramic selected from oxy apatite, tricalcium phosphate (TCP) and porcine bone powder.
- PLLA poly (L-lactic acid)
- PLLA poly (D, L-lactic acid)
- PGA poly (glycolic acid)
- PCL poly (caprolactone)
- Synthesis of three-dimensional pore structure includes particle leaching, gas foaming, fiber meshes, phase separation, emulsion freeze drying, etc.
- this synthesis method is not easy to control the size of the pores, the surface area and porosity of the obtained support is relatively low, there is a problem such that the pore clogging phenomenon of the surface of the support is caused because the open structure is not well formed between the pores.
- a rapid prototyping method has been proposed for the creation of a scaffold with the aid of a computer, and this method solves the above problems and increases the pore size (giant pores) required for cell growth. 1000 ⁇ m) is effective to produce three-dimensional.
- Scaffolds to which the cells of the present invention are immobilized are manufactured by rapid prototyping by mixing biodegradable ceramics with biocompatible or biodegradable polymers to increase mechanical strength while maintaining biocompatibility.
- the nanofiber sheet 20 is made by using a biocompatible or biodegradable polymer solution to create a similar environment in vivo to the cells to be cultured.
- a biocompatible or biodegradable polymer solution to create a similar environment in vivo to the cells to be cultured.
- poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL), poly (hydroxyalkanoate ), Polydioxanone (PDS), polytrimethylene carbonate, derivatives and copolymers thereof are prepared by electrospinning a biocompatible or biodegradable polymer solution selected from the group consisting of.
- a general electrospinning device capable of applying a high voltage (5 to 50 kV) may be used as the electrospinning device used for electrospinning a biocompatible or biodegradable polymer solution.
- the applied voltage range during radiation is preferably performed at 5 to 35 kV, more preferably 15 to 25 kV.
- the distance between the spinneret and the integrated plate is 5 to 30 cm, more preferably 10 to 15 cm.
- Spinning time is preferably 2 to 6 hours.
- Such a scaffold assembly may be used by stacking a plurality of scaffold assemblies.
- 2 illustrates a state and a side view of the plurality of scaffold assemblies stacked.
- FIGS. 3 and 4 show a perfusion bioreactor according to the present invention.
- the scaffold assembly accommodating portion 230 is formed in a groove shape, and the scaffold assembly accommodating portion 230 is formed in any of claims 1 to 5.
- a scaffold assembly 200 for in vitro growth of hematopoietic stem or progenitor cells which is fixed to each scaffold constituting the scaffold assembly (not shown); And a liquid contained for carrying oxygen.
- the cells cultured and differentiated by the perfusion bioreactor according to the present invention may be hematopoietic stem cells or progenitor cells, but are not particularly limited.
- the perfusion bioreactor further includes an oxygen transport liquid inlet, an oxygen transport liquid outlet, and a receptacle for receiving the scaffold assembly.
- the scaffold assembly 200 of the present invention is characterized in that it is accommodated in a sliding manner in the scaffold assembly receiving portion 230 after the plurality is stacked.
- the scaffold assembly receiving portion 230 is characterized in that the internal concave-convex structure for fixing the scaffold assembly.
- the scaffold assembly which is stacked in plural numbers without a separate fixing device by the internal concave-convex structure prevents the scaffold assembly from being separated or distorted from the percussion stimulus by a pump without a separate adhesive. .
- Bioreaction system for in vitro growth of hematopoietic stem or progenitor cells according to the present invention comprises a perfusion type bioreactor 200; A pump 300 connected with the perfusion bioreactor to create a flow having constant vibration in the perfusion bioreactor; A culture solution supply unit 400 for supplying a culture solution to the perfusion type bioreactor; And a control unit 500 for controlling the culture medium supply rate and the number of vibrations applied into the perfusion type bioreactor.
- the scaffold assembly of the present invention and the perfusion reactor using the same are made of materials having biocompatibility, chemical compatibility and mechanical compatibility, and are three-dimensional for cell growth by rapid prototyping. Composed of nanosheets and a scaffold that provides an environmental environment, it is structurally similar to an osteoblastic niche, and a certain size of percussion is pumped by the pump without losing the cells fixed to the scaffold. It is possible that the cells immobilized on the scaffold are exposed to a constant stimulus to provide an optimal environment similar to that in vivo for in vitro growth of hematopoietic stem or progenitor cells.
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Abstract
The present invention relates to a scaffold assembly for in vitro expansion of haematopoietic stem cells or precursor cells, and to a perfusion bioreactor and bioreaction system for in vitro expansion of haematopoietic stem cells or precursor cells using the scaffold assembly. The scaffold assembly and the perfusion reactor using same of the present invention use materials having biocompatibility, chemical compatibility and mechanical compatibility, and comprise nanosheets and a scaffold providing a three dimensional environment for cell growth by means of rapid prototyping, and thus not only is there structural similarity to an osteoblastic niche but also the cells immobilized on the scaffold are not washed away and, at the same time, it is possible to percussion tap to a constant magnitude by means of a pump, and the cells immobilized on the scaffold are exposed to a constant stimulus such that an optimal environment similar to an in vivo environment is provided for the in vitro growth of the haematopoietic stem cells or precursor cells.
Description
본 발명은 조혈모세포 또는 전구 세포의 체외 확대 성장을 위한 스캐폴드 어셈블리, 상기 스캐폴드 어셈블리를 이용하는 조혈모세포 또는 전구 세포의 체외 확대 성장을 위한 관류형 생물 반응기 및 생물 반응 시스템에 관한 것이다.The present invention relates to a scaffold assembly for in vitro expansion of hematopoietic stem or progenitor cells, a perfusion bioreactor and a bioreaction system for in vitro expansion of hematopoietic stem or progenitor cells using the scaffold assembly.
조혈모세포 (hematopoietic stem cell)는 혈액을 구성하는 적혈구, 백혈구 및 혈소판 등의 혈액세포로 분화할 수 있는 능력을 지닌 세포를 말한다. 조혈모세포는 특히 골수에서 대량으로 생산되며, 정상인의 골수 세포 중 약 1% 가량 존재한다. 줄기세포(stem cell)의 특징인 자가복제능력(self-renewing capacity), 다세포분열능(high capacity for cell division), 다분화잠재능(multi-potential differentiation capacity)등을 지니고 있다(van der Kooy D 등 2000, science, 287 ; 1439-1441).Hematopoietic stem cells refer to cells having the ability to differentiate into blood cells such as red blood cells, white blood cells, and platelets, which constitute blood. Hematopoietic stem cells are produced in large quantities especially in the bone marrow and present in about 1% of normal bone marrow cells. It has self-renewing capacity, high capacity for cell division, and multi-potential differentiation capacity that are characteristic of stem cells (van der Kooy D, etc.). 2000, science, 287; 1439-1441).
그러나 재생불량성빈혈 (aplastic anemia)처럼 조혈모세포의 수가 부족한 경우, 백혈병 (leukemia), 골수이형성 증후군 (myelodysplastic syndrome) 등과 같이 조혈모세포가 병든 경우, 항암제 투여 또는 방사선 조사 등으로 조혈모세포가 손상된 경우에는 조혈모세포가 제대로 기능을 발휘할 수 없어 혈액세포를 만들어 낼 수 없게 된다. However, if the number of hematopoietic stem cells is insufficient, such as aplastic anemia, if hematopoietic stem cells are sick, such as leukemia, myelodysplastic syndrome, etc. The parent cell cannot function properly and cannot produce blood cells.
이러한 경우 환자의 조혈모세포를 완전히 제거한 후 정상적인 조혈모세포를 환자에게 투여하면 조혈모세포는 환자의 골수로 이동하여 생착한 후 정상 혈액세포를 다시 생산함으로써 정상적인 조혈기능을 회복하게 되는데, 이를 조혈모세포 이식 (hematopoietic stem cell transplantation)이라 한다. 조혈모세포를 제공할 수 있는 원천 재료로는 골수, 제대혈, 말초혈 등이 있고, 각각을 이용하여 조혈모세포이식을 시행할 경우, 각각을 골수이식, 제대혈이식, 말초혈이식이라고 부르기도 하며, 환자의 상태와 공여자의 가능 여부 등에 따라 종류를 결정하게 된다.In this case, if the hematopoietic stem cells are completely removed and the normal hematopoietic stem cells are administered to the patient, the hematopoietic stem cells migrate to the patient's bone marrow and regenerate normal blood cells to restore normal hematopoietic function. It is called hematopoietic stem cell transplantation. Source materials that can provide hematopoietic stem cells include bone marrow, umbilical cord blood, and peripheral blood. When hematopoietic stem cell transplantation is performed using each of them, each is referred to as bone marrow transplantation, cord blood transplantation, and peripheral blood transplantation. The type will depend on the condition of the donor and the availability of the donor.
그러나, 이러한 조혈모세포는 인간의 몸에선 골수(bone marrow)에 아주 조금 존재하고 있는 세포이기도 하다. 따라서, 조혈모세포 이식을 위해서는 우리가 필요로 하는 조혈모세포를 대량으로 얻는 기술의 개발이 매우 유용하다. However, these hematopoietic stem cells are also very small cells in the bone marrow in the human body. Therefore, the development of a technique for obtaining a large amount of hematopoietic stem cells that we need for hematopoietic stem cell transplantation is very useful.
그간 이러한 조혈모세포를 대량으로 얻기 위한 여러 가지 방법들이 시도되어져 왔었으며, 대표적으로 체외(ex vivo)에서 여러 가지 사이토카인(cytokine)들을 배지에 첨가하여 배양(culture)하는 방법들이 있었다(D Metcalf, 2001, Biomed Pharmacother, 55 ; 75-78, Ian McNiece등, 2001, Experimental Hematology, 29 ; 3-11).Various methods have been attempted to obtain such hematopoietic stem cells in large quantities, and there have been various methods of adding various cytokines to the culture medium in vitro (D Metcalf, 2001, Biomed Pharmacother, 55; 75-78, Ian McNiece et al., 2001, Experimental Hematology, 29; 3-11).
예를 들면, 제대혈 유래 조혈모세포와 조혈전구세포 (hematopoietic progenitor cells, HPCs)들은, 재조합 싸이토카인이 첨가된 상태에서 세포영양막(cell feeder layer)과 함께 공동배양하면 충분히 체외 증식이 가능하다는 보고들이 있다[Slovick FT, et al. (1984) Exp Hematol 12: 327-338 ; Kohler T, et al. (1999) Stem Cells 17: 19-24; McNiece IK, et al. (2002) Exp Hematol 30: 612-616]. 이러한 목적에 사용된 재조합 싸이토카인들로 Flk-2/Flt-3 ligand (FL), stem cell factor (SCF), thrombopoietin (TPO), granulocyte-colony stimulating factor (G-CSF), megakaryocyte growth and development factor (MGDF), interleukin-3 (IL-3), and interleukin-6 (IL-6)이 있다 [Petzer AL, et al. (1996) J Exp Med 183: 2551-2558; McNiece I, et al. (2000) Exp Hematol 28: 1181-1186; Conneally E, et al.(1997) Proc Natl Acad Sci U S A 94: 9836-9841; Ueda T, et al. (2000) J Clin Invest 105: 1013-1021].For example, cord blood-derived hematopoietic stem cells and hematopoietic progenitor cells (HPCs) have been reported to be capable of sufficient in vitro growth when co-cultured with a cell feeder layer in the presence of recombinant cytokines. Slovick FT, et al. (1984) Exp Hematol 12: 327-338; Kohler T, et al. (1999) Stem Cells 17: 19-24; McNiece IK, et al. (2002) Exp Hematol 30: 612-616. Recombinant cytokines used for this purpose include Flk-2 / Flt-3 ligand (FL), stem cell factor (SCF), thrombopoietin (TPO), granulocyte-colony stimulating factor (G-CSF), megakaryocyte growth and development factor ( MGDF), interleukin-3 (IL-3), and interleukin-6 (IL-6) [Petzer AL, et al. (1996) J Exp Med 183: 2551-2558; McNiece I, et al. (2000) Exp Hematol 28: 1181-1186; Conneally E, et al. (1997) Proc Natl Acad Sci U S A 94: 9836-9841; Ueda T, et al. (2000) J Clin Invest 105: 1013-1021.
또한 상기 방법들에 더하여, 세포의 생존(survival)을 증가시키는 신호 전달 물질을 첨가시키거나(Bhardwaj 등, 2001, Nature Immun., 2; 172-180), 기질세포(stroma cell)를 이용하여 직접적인 세포 사이의 상호작용(cell-to-cell interaction)을 통하여 세포증식에 도움을 줄 수 있는 공급세포(feeder cell)들을 사용하는 방법도 고안된 바 있다(Shimakura 등, 2000, Stem Cells, 18; 183-189).In addition to the above methods, it is also possible to add signal transduction agents that increase the survival of the cells (Bhardwaj et al., 2001, Nature Immun., 2; 172-180) or directly using stroma cells. Methods have also been designed to use feeder cells that can help cell proliferation through cell-to-cell interactions (Shimakura et al., 2000, Stem Cells, 18; 183- 18). 189).
전술한 바와 같이 다양한 조혈모세포의 배양방법이 알려져 있으나, 조혈모세포는 체외(ex vivo)에서의 배양시 많은 세포들이 세포사멸을 통하여 손실된다. 더욱이 채취된 조혈모세포의 보관을 위해 조혈모세포를 냉동시킨 후 다시 해동할 경우, 더 많은 조혈모세포들이 세포사멸을 겪게 됨도 확인하였다. 이로 인해 기존의 방법으로는 조혈모세포를 7일 동안 30 내지 40배 정도밖에는 증식하지 못하는 한계가 있었다.As described above, various methods of culturing hematopoietic stem cells are known, but hematopoietic stem cells are lost through apoptosis when cultured in vitro. In addition, it was also confirmed that if hematopoietic stem cells were frozen and thawed again for storage of the collected hematopoietic stem cells, more hematopoietic stem cells would undergo cell death. Due to this, the conventional method has a limit of proliferating hematopoietic stem cells only about 30 to 40 times in 7 days.
본 발명은 상기와 같은 과제를 해결하기 위한 것으로서, 골모세포성 니치(osteoblastic niche)와 유사한 구조의 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리를 제공하는 것을 목적으로 한다. An object of the present invention is to provide a scaffold assembly for in vitro growth of hematopoietic stem cells or progenitor cells of a structure similar to osteoblastic niche.
본 발명은 또한, 체내의 환경과 유사한 환경을 제공하기 위한 상기 스캐폴드 어셈블리를 이용하는 조혈모세포 또는 전구 세포의 체외 성장을 위한 관류형 생물 반응기 및 생물 반응 시스템을 제공하는 것을 목적으로 한다. The present invention also aims to provide a perfusion bioreactor and bioreaction system for in vitro growth of hematopoietic stem or progenitor cells using the scaffold assembly to provide an environment similar to the environment in the body.
본 발명의 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리는 하나 또는 복수개의 나노 파이버 시트; 및 상기 나노 파이버 시트 사이에 적층된 하나 또는 복수개의 격자형 스캐폴드를 포함한다. The scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells of the present invention comprises one or a plurality of nanofiber sheets; And one or a plurality of lattice scaffolds stacked between the nanofiber sheets.
본 발명의 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리에 있어서, 상기 나노 파이버 시트는 전기 방사법에 의하여 제조되는 것을 특징으로 한다. In the scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells of the present invention, the nanofiber sheet is characterized in that it is produced by electrospinning.
본 발명의 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리에 있어서, 상기 나노 파이버 시트는 폴리(L-락트산)(PLLA), 폴리(D,L-락트산)(PDLLA), 폴리(글리콜산)(PGA), 폴리(카프로락톤)(PCL), 폴리(하이드록시알카노에이트), 폴리다이옥산온(PDS), 폴리트라이메틸린카보네이트, 이들의 유도체 및 공중합체로 구성된 군으로부터 선택되는 생체적 적합성 또는 생분해성 고분자로 이루어지는 것을 특징으로 한다. In the scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells of the present invention, the nanofiber sheet is made of poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid). ) (PGA), poly (caprolactone) (PCL), poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, derivatives and copolymers thereof It is characterized by consisting of a suitable or biodegradable polymer.
본 발명의 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리에 있어서, 상기 격자 구조의 스캐폴드는 쾌속 조형법에 의하여 제조되는 것을 특징으로 한다. In the scaffold assembly for in vitro growth of hematopoietic stem cells or progenitor cells of the present invention, the lattice structure scaffold is characterized by being produced by rapid prototyping.
본 발명의 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리에 있어서, 상기 격자 구조의 스캐폴드는 폴리(L-락트산)(PLLA), 폴리(D,L-락트산)(PDLLA) , 폴리(글리콜산)(PGA), 폴리(카프로락톤)(PCL), 폴리(하이드록시알카노에이트), 폴리다이옥산온(PDS), 폴리트라이메틸린카보네이트, 이들의 유도체 및 공중합체로 구성된 군으로부터 선택되는 생체적합성 또는 생분해성 고분자와 생체활성유리, 하이드록시 아파타이트, 트리칼슘포스페이트(tricalcium phosphate; TCP) 및 돼지뼈 분말 중에서 선택되는 생분해성 세라믹을 포함하는 것을 특징으로 한다. In the scaffold assembly for the in vitro growth of hematopoietic stem or progenitor cells of the present invention, the lattice scaffolds are poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly ( Glycolic acid) (PGA), poly (caprolactone) (PCL), poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethylenecarbonate, derivatives and copolymers thereof And biodegradable ceramics selected from biocompatible or biodegradable polymers and bioactive glass, hydroxyapatite, tricalcium phosphate (TCP) and porcine bone powder.
본 발명은 또한, 상기 본 발명의 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리; 상기 스캐폴드 어셈블리의 스캐폴드에 고정된 세포; 및 산소를 운반하기 위해 수용된 액체를 포함하는 관류형 생물 반응기를 제공한다. The present invention also comprises a scaffold assembly for in vitro growth of the hematopoietic stem or progenitor cells of the present invention; Cells anchored to a scaffold of the scaffold assembly; And a perfused bioreactor comprising a liquid contained for carrying oxygen.
본 발명의 관류형 생물 반응기는 산소 운반 액체 유입구, 산소 운반 액체 배출구 및 상기 스캐폴드 어셈블리를 수용하기 위한 수용부를 포함하는 것을 특징으로 한다. The perfusion bioreactor of the present invention is characterized in that it comprises an oxygen carrier liquid inlet, an oxygen carrier liquid outlet and a receptacle for receiving the scaffold assembly.
본 발명의 관류형 생물 반응기에 있어서, 상기 스캐폴드 어셈블리는 상기 스캐폴드 어셈블리를 수용하기 위한 수용부는 별도의 접착제나 고정제 없이 상기 스캐폴드 어셈블리 수용부에 슬라이딩 방식으로 수용되는 것을 특징으로 한다. In the perfusion bioreactor of the present invention, the scaffold assembly is characterized in that the receiving portion for receiving the scaffold assembly is accommodated in the scaffold assembly receiving portion in a sliding manner without a separate adhesive or fixing agent.
본 발명의 관류형 생물 반응기에 있어서, 상기 세포는 조혈모세포 또는 전구 세포 인 것을 특징으로 한다. In the perfusion bioreactor of the present invention, the cells are characterized in that hematopoietic stem cells or progenitor cells.
본 발명은 또한, 본 발명에 의한 관류형 생물 반응기; 상기 관류형 생물 반응기와 연결되어 상기 관류형 생물 반응기 내에 일정한 진동을 가진 흐름을 만들기 위한 펌프; 상기 관류형 생물 반응기에 배양액을 공급하기 위한 배양액 공급부; 및 상기 관류형 생물 반응기 내부로의 배양액 공급 속도, 진동 인가 횟수를 조절하기 위한 제어부를 포함하는 것인 조혈모세포 또는 전구 세포의 체외 성장을 위한 생물 반응 시스템을 제공한다. The present invention also provides a perfusion bioreactor according to the present invention; A pump connected with the perfusion bioreactor to create a flow with constant vibration in the perfusion bioreactor; A culture medium supply unit for supplying a culture medium to the perfusion type bioreactor; And it provides a biological reaction system for in vitro growth of hematopoietic stem cells or progenitor cells comprising a control unit for controlling the culture medium supply rate, the number of vibrations applied into the perfusion type bioreactor.
본 발명의 조혈모세포 또는 전구 세포의 체외 성장을 위한 생물 반응 시스템은 상기 관류형 생물 반응기로부터 나오는 산소 운반 액체로부터 바이오 매스를 제거하기 위한 바이오매스 제거 장치, 산소 운반 액체의 합성 또는 반-합성 성분을 다른 성분과 분리하기 위한 분리 장치, 산소 운반 액체로부터 이산화탄소를 제거하기 위한 이산화탄소 제거 장치, 산소 운반 액체에 산소를 첨가하는 산소첨가 장치, 및 산소 운반 액체의 공기제거를 위한 공기제거 장치로 이루어진 그룹으로부터 선택된 하나 이상의 장치를 더 포함하는 것을 특징으로 한다.The bioreaction system for in vitro growth of hematopoietic stem or progenitor cells of the present invention comprises a biomass removal device for removing biomass from an oxygen carrier liquid from the perfusion bioreactor, a synthetic or semi-synthetic component of an oxygen carrier liquid. From a group consisting of a separation device for separating carbon dioxide from other components, a carbon dioxide removal device for removing carbon dioxide from an oxygen carrier liquid, an oxygen addition device for adding oxygen to the oxygen carrier liquid, and an air removal device for deaeration of the oxygen carrier liquid. It further comprises one or more selected devices.
본 발명의 스캐폴드 어셈블리 및 이를 이용한 관류형 반응기는 생체 친화성(biocompatibility), 화학적 적합성(chemicalcompatibility) 및 기계적 적합성(mechanical compatibility)을 가지는 재료를 사용하고, 쾌속 조형법에 의하여 세포 성장을 위한 3차원적 환경을 제공하는 스캐폴드와, 나노 시트로 구성됨으로써 구조적으로 골모세포성 니치(osteoblastic niche)와 유사할 뿐만 아니라, 상기 스캐폴드에 고정된 세포가 유실되지 않으면서 펌프에 의하여 일정 크기의 타진이 가능하여 상기 스캐폴드에 고정된 세포가 일정한 자극에 노출되어 조혈모세포 또는 전구 세포의 체외 성장을 위한 생체내와 유사한 최적의 환경을 제공하는 효과가 있다.The scaffold assembly of the present invention and the perfusion reactor using the same are made of materials having biocompatibility, chemical compatibility and mechanical compatibility, and are three-dimensional for cell growth by rapid prototyping. Composed of nanosheets and a scaffold that provides an environmental environment, it is structurally similar to an osteoblastic niche, and a certain size of percussion is pumped by the pump without losing the cells fixed to the scaffold. It is possible that the cells immobilized on the scaffold are exposed to a constant stimulus to provide an optimal environment similar to that in vivo for in vitro growth of hematopoietic stem or progenitor cells.
도 1은 본 발명에 의한 하나의 나노파이버 시트와 하나의 격자형 스캐폴드가 적층된 조혈모세포 또는 전구 세포의 체외 성장을 위한 골모세포성 니치(osteoblastic niche) 스캐폴드 어셈블리를 나타낸다. 1 shows an osteoblastic niche scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells in which one nanofiber sheet and one lattice scaffold are stacked according to the present invention.
도 2는 본 발명에 의한 복수개의 나노파이버 시트와 복수개의 격자형 스캐폴드가 적층된 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리의 사시도 및 측면확대도를 나타낸다. Figure 2 shows a perspective view and a side view of a scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells stacked with a plurality of nanofiber sheets and a plurality of lattice scaffold according to the present invention.
도 3는 본 발명에 의한 조혈모세포 또는 전구 세포의 체외 성장을 위한 관류형 반응기를 나타낸다. Figure 3 shows a perfusion reactor for in vitro growth of hematopoietic stem or progenitor cells according to the present invention.
도 4는 본 발명에 의한 조혈모세포 또는 전구 세포의 체외 성장을 위한 관류형 반응기의 스캐폴드 수용부에 상기 스캐폴드 어셈블리가 수용되는 상태를 나타낸다. Figure 4 shows the state in which the scaffold assembly is accommodated in the scaffold receiving portion of the perfusion reactor for in vitro growth of hematopoietic stem or progenitor cells according to the present invention.
도 5는 본 발명에 의한 조혈모세포 또는 전구 세포의 체외 성장을 위한 생물 반응 시스템을 나타낸다. 5 shows a biological response system for in vitro growth of hematopoietic stem or progenitor cells according to the present invention.
이하, 도면을 참조하여 본 발명을 더욱 상세히 설명한다. Hereinafter, the present invention will be described in more detail with reference to the drawings.
도 1은 본 발명에 의한 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리(100) 를 나타낸다. 도 1에서 상기 스캐폴드 어셈블리(100)는 하나의 나노파이버 시트(20)와 하나의 격자형 스캐폴드(10)가 순차적으로 적층되어 구성된다. 1 illustrates a scaffold assembly 100 for in vitro growth of hematopoietic stem or progenitor cells according to the present invention. In FIG. 1, the scaffold assembly 100 is formed by sequentially stacking one nanofiber sheet 20 and one lattice scaffold 10.
본 발명에 의한 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리(100)는 하나의 나노파이버 시트(20)와 하나의 격자형 스캐폴드(10)가 순차적으로 적층되어 조혈모세포의 생장을 위해 체내에서의 환경과 유사한 골모세포성 니치(osteoblastic niche) 구조를 형성한다. In the scaffold assembly 100 for in vitro growth of hematopoietic stem or progenitor cells according to the present invention, one nanofiber sheet 20 and one lattice-type scaffold 10 are sequentially stacked to grow hematopoietic stem cells. It forms an osteoblastic niche structure similar to the environment in the body.
조혈모세포 니치는 조혈모세포의 이동성과 휴면 및 분화를 조절하고 조혈모세포의 유지와 자생을 증진시키는 세포막결합 단백질 혹은 분비 인자를 발현시키는 지지 세포에 의해 만들어지는 특수화된 미세 환경을 의미하며, 이러한 조혈모세포 니치에 대한 개념은 1978년 schofield 에 의해 도입되었다. 조혈모세포 니치는 대표적으로 골모세포성 니치(osteoblastic niche)과 혈관성 니치(vascular niche)가 알려져 있으며, 본원 발명의 상기 격자형 스캐폴드(10)는 상기 골모세포성 니치(osteoblastic niche) 역할을 하여 조혈모세포 생장에 적합한 환경을 제공한다. Hematopoietic stem cell niche refers to a specialized microenvironment made by supportive cells expressing cell membrane-binding proteins or secretory factors that regulate the mobility, dormancy and differentiation of hematopoietic stem cells, and promote maintenance and autologous hematopoietic stem cells. The concept of niches was introduced by schofield in 1978. Hematopoietic stem cell niches are typically known as osteoblastic niche and vascular niche, and the lattice scaffold 10 of the present invention serves as the osteoblastic niche and is a hematopoietic Provide an environment suitable for blast growth.
본 발명의 상기 스캐폴드 어셈블리(100)에 있어서, 상기 격자형 스캐폴드(10)는 쾌속 조형법으로 형성되어 조혈모세포의 생장에 유리한 미세 환경(niche)를 만들어 주어 골조직 재생을 위하여 세포가 고정화되는 지지대 역할을 한다. In the scaffold assembly 100 of the present invention, the lattice-type scaffold 10 is formed by a rapid molding method to create a micro environment (niche) favorable for the growth of hematopoietic stem cells to immobilize the cells for bone tissue regeneration Serve as a support
본 발명의 상기 스캐폴드 어셈블리(100)에 있어서, 상기 나노파이버 시트(20)는 상기 격자형 스캐폴드에 적층된 후 스캐폴드 어셈블리 수용부에 수용되어, 펌프를 구비한 관류형 반응기에 의하여 주기적이고 지속적인 펌프에 의한 타진 자극을 받으면서 세포가 배양되는 동안 타진에 의하여 세포가 휩쓸려 가지 못하도록 막아주는 장벽 역할 뿐만 아니라, 표면적이 매우 크기 때문에 조혈모세포 성장을 위한 성장 인자 등의 로딩이 용이하다는 장점을 나타내게 된다. In the scaffold assembly 100 of the present invention, the nanofiber sheet 20 is laminated to the lattice scaffold and then accommodated in the scaffold assembly receiving portion, and is periodically circulated by a perfusion reactor with a pump. The percussive stimulation of percussion by a continuous pump not only acts as a barrier to prevent the cells from being swept by the percussion while the cell is incubated, but also shows the advantage of easy loading of growth factors for hematopoietic stem cell growth due to its large surface area. .
본 발명의 스캐폴드 어셈블리는 나노 사이즈 기공과 마이크로 사이즈 기공이 모두 존재하여 조혈모세포 성장을 위한 최적의 환경을 제공한다. 즉, 상기 나노 파이버 시트는 1 내지 100 nm 범위의 나노 사이즈 기공이 3차원적으로 상호 연결된 형태로 형성되며, 상기 격자형 스캐폴드는 100 내지 1000 ㎛ 범위의 기공이 3차원으로 상호 연결된 형태로 형성되어, 본 발명의 스캐폴드 어셈블리 내에 생체 환경과 유사하도록 다양한 크기의 기공이 포함됨으로써, 개개 세포에 영향분과 산소를 충분히 공급하면서도 펌프에 의한 타진 자극이 골고루 전달되어 효율적인 세포 증식 및 분화, 세포 괴사 방지 등의 효과를 나타내도록 한다. The scaffold assembly of the present invention has both nano size pores and micro size pores to provide an optimal environment for hematopoietic stem cell growth. That is, the nanofiber sheet is formed in the form of three-dimensional interconnected nano-size pores in the range of 1 to 100 nm, the lattice scaffold is formed in the form of three-dimensional interconnected pores in the range of 100 to 1000 ㎛ The pores of various sizes are included in the scaffold assembly of the present invention to be similar to the living environment, and the percussion stimulus by the pump is evenly delivered while supplying the affected substances and oxygen to the individual cells efficiently, thereby preventing efficient cell proliferation and differentiation and cell necrosis. To effect such as.
상기 격자 구조의 스캐폴드(10)는 폴리(L-락트산)(PLLA), 폴리(D,L-락트산)(PDLLA) , 폴리(글리콜산)(PGA), 폴리(카프로락톤)(PCL), 폴리(하이드록시알카노에이트), 폴리다이옥산온(PDS), 폴리트라이메틸린카보네이트, 이들의 유도체 및 공중합체로 구성된 군으로부터 최소 1 종 이상 선택되는 생체적합성 또는 생분해성 고분자와 생체활성유리, 하이드록시 아파타이트, 트리칼슘포스페이트(tricalcium phosphate; TCP) 및 돼지뼈 분말 중에서 선택되는 생분해성 세라믹을 포함하는 재료를 사용하여 3차원 기공 구조를 가지도록 제조된다. The lattice scaffold 10 includes poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL), At least one or more biocompatible or biodegradable polymers selected from the group consisting of poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethyl carbonate, derivatives and copolymers thereof, It is prepared to have a three-dimensional pore structure using a material comprising a biodegradable ceramic selected from oxy apatite, tricalcium phosphate (TCP) and porcine bone powder.
3차원 기공구조의 합성법으로는 입자 침출(particle leaching)법, 가스를 이용한 거품성형 (gas foaming)법, 섬유망사 (fiber meshes)법, 상분리 (phase separation)법, 동결유탁 (emulsion freeze drying) 등이 있으나 이러한 합성방법은 기공의 크기를 조절하는 것이 쉽지 않고 얻어지는 지지체의 표면적과 기공률이 비교적 낮으며 기공 간 열린 구조가 잘 형성되지 않아 지지체 표면의 기공 막힘 현상이 야기되는 등의 문제가 있었다. 최근에는 컴퓨터의 도움을 받아 실체의 모델을 만드는 쾌속조형 (rapid prototyping)법이 지지체의 제작에 제안되어지고 있으며 이 방법은 위의 문제를 해결하고 세포의 성장에 필요한 기공크기 (자이언트기공; 100~1000μm)를 3차원적으로 제작하는데 효율적이다. 본 발명의 세포가 고정화되는 스캐폴드는 생체적합성을 유지하면서도 기계적 강도를 높일 수 있도록 생체적합성 또는 생분해성 고분자에 생분해성 세라믹을 혼합하여 쾌속 조형법으로 제조된다.Synthesis of three-dimensional pore structure includes particle leaching, gas foaming, fiber meshes, phase separation, emulsion freeze drying, etc. However, this synthesis method is not easy to control the size of the pores, the surface area and porosity of the obtained support is relatively low, there is a problem such that the pore clogging phenomenon of the surface of the support is caused because the open structure is not well formed between the pores. Recently, a rapid prototyping method has been proposed for the creation of a scaffold with the aid of a computer, and this method solves the above problems and increases the pore size (giant pores) required for cell growth. 1000μm) is effective to produce three-dimensional. Scaffolds to which the cells of the present invention are immobilized are manufactured by rapid prototyping by mixing biodegradable ceramics with biocompatible or biodegradable polymers to increase mechanical strength while maintaining biocompatibility.
본 발명에서 상기 나노 파이버 시트(20)는 생체적 적합성 또는 생분해성 고분자 용액을 사용하여 제조함으로써 배양되는 세포에게 생체 내 환경과 유사한 환경을 만들어 주도록 한다. 구체적으로는 폴리(L-락트산)(PLLA), 폴리(D,L-락트산)(PDLLA), 폴리(글리콜산)(PGA), 폴리(카프로락톤)(PCL), 폴리(하이드록시알카노에이트), 폴리다이옥산온(PDS), 폴리트라이메틸린카보네이트, 이들의 유도체 및 공중합체로 구성된 군으로부터 선택되는 생체적 적합성 또는 생분해성 고분자 용액을 전기 방사하여 제조된다.In the present invention, the nanofiber sheet 20 is made by using a biocompatible or biodegradable polymer solution to create a similar environment in vivo to the cells to be cultured. Specifically, poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL), poly (hydroxyalkanoate ), Polydioxanone (PDS), polytrimethylene carbonate, derivatives and copolymers thereof are prepared by electrospinning a biocompatible or biodegradable polymer solution selected from the group consisting of.
전기 방사(ELECTROSPINNING)를 이용하여 그물망 나노파이버를 형성하는 기술은 널리 알려져 있다. 본 발명에서는 생체적 적합성 또는 생분해성 고분자 용액을 전기 방사하기 위하여 사용하는 전기 방사 장치로는 고전압(5 내지 50 kV)을 걸어줄 수 있는 일반적인 전기 방사 장치가 사용될 수 있다. 방사시의 인가전압 범위는 5 내지 35kV에서 수행하는 것이 바람직하며, 더욱 바람직하게는 15 내지 25 kV이다. 방사구와 집적판 사이의 거리는 5 내지 30cm이며, 더욱 바람직하게는 10 내지 15cm이다. 방사 시간은 2 내지 6시간이 바람직하다.Techniques for forming mesh nanofibers using electrospinning are well known. In the present invention, a general electrospinning device capable of applying a high voltage (5 to 50 kV) may be used as the electrospinning device used for electrospinning a biocompatible or biodegradable polymer solution. The applied voltage range during radiation is preferably performed at 5 to 35 kV, more preferably 15 to 25 kV. The distance between the spinneret and the integrated plate is 5 to 30 cm, more preferably 10 to 15 cm. Spinning time is preferably 2 to 6 hours.
이와 같은 스캐폴드 어셈블리는 복수개가 적층되어 사용될 수 있다. 도 2에 상기 스캐폴드 어셈블리 복수개가 적층된 상태 및 측면도를 나타내었다. Such a scaffold assembly may be used by stacking a plurality of scaffold assemblies. 2 illustrates a state and a side view of the plurality of scaffold assemblies stacked.
도 3, 도 4 에 본 발명에 따르는 관류형 생물 반응기를 나타내었다. 도 3, 도 4에서 보는 바와 같이 본 발명에 따르는 관류형 생물 반응기는 스캐폴드 어셈블리 수용부(230)가 홈 형태로 형성되고, 상기 스캐폴드 어셈블리 수용부(230)에 제 1항 내지 제 5항의 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리(200)가 수용되며, 상기 스캐폴드 어셈블리를 구성하는 각각의 스캐폴드에 고정된 세포(미도시); 및 산소를 운반하기 위해 수용된 액체를 포함한다. 본 발명에 따르는 관류형 생물 반응기에 의하여 배양 및 분화되는 세포는 조혈모세포 또는 전구 세포일 수 있지만, 특별히 제한되지는 않는다. 3 and 4 show a perfusion bioreactor according to the present invention. As shown in FIGS. 3 and 4, in the perfusion type bioreactor according to the present invention, the scaffold assembly accommodating portion 230 is formed in a groove shape, and the scaffold assembly accommodating portion 230 is formed in any of claims 1 to 5. A scaffold assembly 200 for in vitro growth of hematopoietic stem or progenitor cells, which is fixed to each scaffold constituting the scaffold assembly (not shown); And a liquid contained for carrying oxygen. The cells cultured and differentiated by the perfusion bioreactor according to the present invention may be hematopoietic stem cells or progenitor cells, but are not particularly limited.
도 3, 도 4에서 보는 바와 같이 상기 관류형 생물 반응기는 산소 운반 액체 유입구, 산소 운반 액체 배출구 및 상기 스캐폴드 어셈블리를 수용하기 위한 수용부를 더 포함하는 것을 특징으로 한다. As shown in FIGS. 3 and 4, the perfusion bioreactor further includes an oxygen transport liquid inlet, an oxygen transport liquid outlet, and a receptacle for receiving the scaffold assembly.
도 4에서 보는 바와 같이 본 발명의 상기 스캐폴드 어셈블리(200)는 복수개가 적층된 후 상기 스캐폴드 어셈블리 수용부(230)에 슬라이딩 방식으로 수용되는 것을 특징으로 한다. 상기 스캐폴드 어셈블리 수용부(230)는 상기 스캐폴드 어셈블리를 고정하기 위한 내부 요철 구조로 되어 있는 것을 특징으로 한다. 본 발명에 있어서는 이와 같은 내부 요철 구조에 의하여 별도의 고정 장치 없이 복수개로 적층되는 스캐폴드 어셈블리 상호가 별도의 접착제가 없이도 상기 스캐폴드 어셈블리가 펌프에 의한 타진 자극에도 분리되거나 형태가 흐트러지는 것을 막게 된다. As shown in Figure 4, the scaffold assembly 200 of the present invention is characterized in that it is accommodated in a sliding manner in the scaffold assembly receiving portion 230 after the plurality is stacked. The scaffold assembly receiving portion 230 is characterized in that the internal concave-convex structure for fixing the scaffold assembly. In the present invention, the scaffold assembly which is stacked in plural numbers without a separate fixing device by the internal concave-convex structure prevents the scaffold assembly from being separated or distorted from the percussion stimulus by a pump without a separate adhesive. .
도 5에 본 발명에 의한 조혈모세포 또는 전구 세포의 체외 성장을 위한 생물 반응 시스템의 개략도를 나타내었다. 본 발명에 의한 조혈모세포 또는 전구 세포의 체외 성장을 위한 생물 반응 시스템은 관류형 생물 반응기(200); 상기 관류형 생물 반응기와 연결되어 상기 관류형 생물 반응기 내에 일정한 진동을 가진 흐름을 만들기 위한 펌프(300); 상기 관류형 생물 반응기에 배양액을 공급하기 위한 배양액 공급부(400); 및 상기 관류형 생물 반응기 내부로의 배양액 공급 속도, 진동 인가 횟수를 조절하기 위한 제어부(500)를 포함한다. 5 shows a schematic diagram of a biological response system for in vitro growth of hematopoietic stem or progenitor cells according to the present invention. Bioreaction system for in vitro growth of hematopoietic stem or progenitor cells according to the present invention comprises a perfusion type bioreactor 200; A pump 300 connected with the perfusion bioreactor to create a flow having constant vibration in the perfusion bioreactor; A culture solution supply unit 400 for supplying a culture solution to the perfusion type bioreactor; And a control unit 500 for controlling the culture medium supply rate and the number of vibrations applied into the perfusion type bioreactor.
본 발명의 스캐폴드 어셈블리 및 이를 이용한 관류형 반응기는 생체 친화성(biocompatibility), 화학적 적합성(chemicalcompatibility) 및 기계적 적합성(mechanical compatibility)을 가지는 재료를 사용하고, 쾌속 조형법에 의하여 세포 성장을 위한 3차원적 환경을 제공하는 스캐폴드와, 나노 시트로 구성됨으로써 구조적으로 골모세포성 니치(osteoblastic niche)와 유사할 뿐만 아니라, 상기 스캐폴드에 고정된 세포가 유실되지 않으면서 펌프에 의하여 일정 크기의 타진이 가능하여 상기 스캐폴드에 고정된 세포가 일정한 자극에 노출되어 조혈모세포 또는 전구 세포의 체외 성장을 위한 생체내와 유사한 최적의 환경을 제공하는 효과가 있다.The scaffold assembly of the present invention and the perfusion reactor using the same are made of materials having biocompatibility, chemical compatibility and mechanical compatibility, and are three-dimensional for cell growth by rapid prototyping. Composed of nanosheets and a scaffold that provides an environmental environment, it is structurally similar to an osteoblastic niche, and a certain size of percussion is pumped by the pump without losing the cells fixed to the scaffold. It is possible that the cells immobilized on the scaffold are exposed to a constant stimulus to provide an optimal environment similar to that in vivo for in vitro growth of hematopoietic stem or progenitor cells.
Claims (11)
- 하나 또는 복수개의 나노 파이버 시트; 및One or a plurality of nanofiber sheets; And상기 나노 파이버 시트 사이에 적층된 하나 또는 복수개의 스캐폴드를 포함하는 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리.A scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells comprising one or a plurality of scaffolds stacked between the nanofiber sheets.
- 제 1 항에 있어서, The method of claim 1,상기 나노 파이버 시트는 폴리(L-락트산)(PLLA), 폴리(D,L-락트산)(PDLLA), 폴리(글리콜산)(PGA), 폴리(카프로락톤)(PCL), 폴리(하이드록시알카노에이트), 폴리다이옥산온(PDS), 폴리트라이메틸린카보네이트, 이들의 유도체 및 공중합체로 구성된 군으로부터 선택되는 생체적 적합성 또는 생분해성 고분자로 이루어지는 것인 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리.The nanofiber sheet is poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL), poly (hydroxyal) Carnoate), polydioxanone (PDS), polytrimethyllincarbonate, derivatives and copolymers thereof for the in vitro growth of hematopoietic stem or progenitor cells consisting of biocompatible or biodegradable polymers selected from the group consisting of: Scaffold assembly.
- 제 2 항에 있어서,The method of claim 2,상기 나노 파이버 시트는 상기 생체적 적합성 또는 생분해성 고분자 용액을 전기 방사하여 제조되는 것인 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리.The nanofiber sheet is a scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells that are prepared by electrospinning the biocompatible or biodegradable polymer solution.
- 제 1 항에 있어서,The method of claim 1,상기 격자 구조의 스캐폴드는 폴리(L-락트산)(PLLA), 폴리(D,L-락트산)(PDLLA) , 폴리(글리콜산)(PGA), 폴리(카프로락톤)(PCL), 폴리(하이드록시알카노에이트), 폴리다이옥산온(PDS), 폴리트라이메틸린카보네이트, 이들의 유도체 및 공중합체로 구성된 군으로부터 선택되는 생체적합성 또는 생분해성 고분자와 The lattice scaffolds include poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL), poly (hydr Oxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, derivatives and copolymers thereof, and a biocompatible or biodegradable polymer selected from the group consisting of하이드록시 아파타이트 또는 돼지뼈 분말 중에서 선택되는 생체적합성 세라믹을 포함하는 것인 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리.A scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells comprising a biocompatible ceramic selected from hydroxyapatite or porcine bone powder.
- 제 4 항에 있어서,The method of claim 4, wherein상기 격자 구조의 스캐폴드는 쾌속 조형법에 의하여 제조되는 것인 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리.The scaffold of the lattice structure is a scaffold assembly for in vitro growth of hematopoietic stem cells or progenitor cells is prepared by rapid prototyping.
- 제 1 항 내지 제 5 항 중 어느 하나의 조혈모세포 또는 전구 세포의 체외 성장을 위한 스캐폴드 어셈블리; A scaffold assembly for in vitro growth of hematopoietic stem or progenitor cells of any one of claims 1 to 5;상기 스캐폴드 어셈블리의 스캐폴드에 고정된 세포; 및 Cells anchored to a scaffold of the scaffold assembly; And산소를 운반하기 위해 수용된 액체;를 포함하는 관류형 생물 반응기.A perfusion bioreactor comprising a liquid contained to carry oxygen.
- 제 6 항에 있어서,The method of claim 6,상기 스캐폴드 어셈블리는 상기 스캐폴드 어셈블리 수용부에 슬라이딩 방식으로 수용되는 것을 특징으로 하는 관류형 생물 반응기.And the scaffold assembly is received in a sliding manner in the scaffold assembly receiving portion.
- 제 6 항에 있어서,The method of claim 6,상기 관류형 생물 반응기는 산소 운반 액체 유입구, 산소 운반 액체 배출구 및 상기 스캐폴드 어셈블리를 수용하기 위한 수용부를 더 포함하는 것을 특징으로 하는 관류형 생물 반응기.The perfusion bioreactor further comprises an oxygen carrier liquid inlet, an oxygen carrier liquid outlet and a receptacle for receiving the scaffold assembly.
- 제 6 항에 있어서,The method of claim 6,상기 세포는 조혈모세포 또는 전구 세포 인 것을 특징으로 하는 관류형 생물 반응기.The cell is a perfusion bioreactor, characterized in that hematopoietic stem cells or progenitor cells.
- 제 6 항 내지 제 9 항 중 어느 하나의 관류형 생물 반응기;10. A perfusion bioreactor according to any one of claims 6 to 9;상기 관류형 생물 반응기와 연결되어 상기 관류형 생물 반응기 내에 일정한 진동을 가진 흐름을 만들기 위한 펌프;A pump connected with the perfusion bioreactor to create a flow with constant vibration in the perfusion bioreactor;상기 관류형 생물 반응기에 배양액을 공급하기 위한 배양액 공급부; 및 A culture medium supply unit for supplying a culture medium to the perfusion type bioreactor; And상기 관류형 생물 반응기 내부로의 배양액 공급 속도, 진동 인가 횟수를 조절하기 위한 제어부를 포함하는 것인 조혈모세포 또는 전구 세포의 체외 성장을 위한 생물 반응 시스템.Bioreaction system for in vitro growth of hematopoietic stem or progenitor cells comprising a control unit for adjusting the culture medium supply rate, the number of vibrations applied into the perfusion type bioreactor.
- 제 10 항에 있어서,The method of claim 10,상기 조혈모세포 또는 전구 세포의 체외 성장을 위한 생물 반응 시스템은 상기 관류형 생물 반응기로부터 나오는 산소 운반 액체로부터 바이오매스를 제거하기 위한 바이오매스 제거 장치, 산소 운반 액체의 합성 또는 반-합성 성분을 다른 성분과 분리하기 위한 분리 장치, 산소 운반 액체로부터 이산화탄소를 제거하기 위한 이산화탄소 제거 장치, 산소 운반 액체에 산소를 첨가하는 산소첨가 장치, 및 산소 운반 액체의 공기제거를 위한 공기제거 장치로 이루어진 그룹으로부터 선택된 하나 이상의 장치를 더 포함하는 것인 조혈모세포 또는 전구 세포의 체외 성장을 위한 생물 반응 시스템.The bioreaction system for in vitro growth of the hematopoietic stem or progenitor cells comprises a biomass removal device for removing biomass from an oxygen carrier liquid from the perfusion bioreactor, a synthetic or semi-synthetic component of the oxygen carrier liquid, and other components. A separation device for separating carbon dioxide from the oxygen carrier liquid, a carbon dioxide removal device for removing carbon dioxide from the oxygen carrier liquid, an oxygen addition device for adding oxygen to the oxygen carrier liquid, and an air removal device for removing air from the oxygen carrier liquid A biological reaction system for in vitro growth of hematopoietic stem or progenitor cells further comprising the above device.
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KR100638736B1 (en) * | 2005-09-16 | 2006-10-25 | 한국기계연구원 | Nano fiber shaped cell culture scaffold and method for preparing the same |
KR20080091827A (en) * | 2006-01-27 | 2008-10-14 | 더 리전트 오브 더 유니버시티 오브 캘리포니아 | Biomimetic scaffolds |
KR100889593B1 (en) * | 2007-02-23 | 2009-03-20 | 부산대학교 산학협력단 | Hybrid Composite of Biopolymers and Bioceramics, and Method for Preparing the Same |
JP2010148496A (en) * | 2008-11-21 | 2010-07-08 | Mitsubishi Rayon Co Ltd | Cell culture scaffold material and cell culture module |
KR20110025327A (en) * | 2009-09-04 | 2011-03-10 | 중앙대학교 산학협력단 | Biphasic scaffold for co-culturing bone cell and cartilage cell |
KR20110028019A (en) * | 2009-09-11 | 2011-03-17 | 연세대학교 산학협력단 | Micropatterned nanofiber scaffold |
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CN103341989A (en) * | 2013-07-08 | 2013-10-09 | 上海大学 | Regeneration bone scaffold forming system and method based on comprehensive 3D printing formation |
CN113736658A (en) * | 2021-11-03 | 2021-12-03 | 北京国卫生物科技有限公司 | Long-term large-scale amplification biological reaction system for human umbilical cord mesenchymal stem cells |
CN113736658B (en) * | 2021-11-03 | 2022-01-25 | 北京国卫生物科技有限公司 | Long-term large-scale amplification biological reaction system for human umbilical cord mesenchymal stem cells |
Also Published As
Publication number | Publication date |
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KR101880676B1 (en) | 2018-07-20 |
WO2013027962A3 (en) | 2013-05-10 |
KR20130021180A (en) | 2013-03-05 |
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