WO2017204518A2 - Induced pluripotent stem cell culture plate using nanotopography and culturing method using same - Google Patents
Induced pluripotent stem cell culture plate using nanotopography and culturing method using same Download PDFInfo
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- WO2017204518A2 WO2017204518A2 PCT/KR2017/005321 KR2017005321W WO2017204518A2 WO 2017204518 A2 WO2017204518 A2 WO 2017204518A2 KR 2017005321 W KR2017005321 W KR 2017005321W WO 2017204518 A2 WO2017204518 A2 WO 2017204518A2
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- the present invention relates to an induced pluripotent stem cell culture plate, and more specifically, by reproducing the effect of extracellular matrix by patterning the surface of the culture plate in nano units, and coating with fibronectin to reduce the contact angle between the culture plate and the cells.
- it while maintaining the stem cell properties, it relates to an induced pluripotent stem cell culture plate capable of obtaining a large amount of induced pluripotent stem cells excellent in proliferation and adhesion ability.
- Stem cells have a proliferative capacity and unique ability to differentiate into various cell types, and provide many alternatives and opportunities in tissue engineering and regenerative medicine. Stem cells exist in useful and tissue specific environments such as complexes of soluble and insoluble factors and regulated biochemical compounds in the body.
- the artificially cultured cells are mainly attached to the bottom of the cell culture vessel to survive the process of growth, proliferation and differentiation, but some cells multiply and multiply on other cells, Some other cells may grow, grow, and differentiate while remaining suspended in cell culture.
- the artificially made cell culture vessel has a surface property different from that of the extracellular matrix in which the cells are originally placed, and thus, cell proliferation and differentiation efficiency may be reduced.
- micro pattern fabrication technology with nanometer precision has been greatly helped by the development of the semiconductor industry in recent years, and various nanometer sizes including on-electron beam technology, scanning probe microscope technology, nano imprint technology, and micro contact printing technology Along with the control technologies of, it is possible to develop application technology using them.
- the pattern can be used as a mold to create an intaglio copy pattern, and this technology can transfer a pattern quickly and easily onto a polymer substrate which is easy to process and chemically modify the surface.
- the present invention has been made to solve the above problems, the present inventors are proliferating and attaching while maintaining stem cellity through a culture membrane and a coating using a modifier on the surface of the membrane and the nano-structure iron and recesses Invented a culture plate for culturing induced pluripotent stem cells with excellent ability.
- a culture membrane in which a convex portion having a diameter of 300-400 nm and a concave portion having a diameter of 100-300 nm are arranged in a plurality to form a pattern;
- Another object of the present invention is to provide a third object of the present invention.
- Induced pluripotent stem cell culture method using induced pluripotent stem cell culture plate comprising the following steps:
- step (d) attaching the induced pluripotent stem cells to the culture table prepared in step (c) to provide a step of culturing.
- a culture membrane in which a convex portion having a diameter of 300-400 nm and a concave portion having a diameter of 100-300 nm are arranged in a plurality to form a pattern;
- It provides an induced pluripotent stem cell culture plate comprising a modifier coated on the culture membrane surface.
- the culture membrane may be made of polydimethylsiloxane (PDMS).
- PDMS polydimethylsiloxane
- the modifier may be Fibronectin.
- the convex portion and the recessed portion may be formed in a hemispherical shape, curved arc shape, wave shape or column shape.
- the culture plate it is possible to improve the adhesion and proliferation of induced pluripotent stem cells (Induced pluripotent stem cells).
- the culture plate it is possible to improve the undifferentiated maintenance of induced pluripotent stem cells (Induced pluripotent stem cells).
- the present invention is a.
- Induced pluripotent stem cell culture method using induced pluripotent stem cell culture plate comprising the following steps:
- step (d) providing a step of culturing by attaching induced pluripotent stem cells to the culture table prepared in step (c).
- Induced pluripotent stem cell culture plate is a patterned culture membrane and a modifier coated on the surface of the culture membrane to reproduce the effects of physical microenvironment, that is, extracellular matrix in vivo using nanotopography
- FIG. 1 is an exploded perspective view of an induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- Figure 2a shows a schematic diagram of a flat pattern membrane (FPM) of the flat form for comparing the effect of the nanotopography (nanotopography) of the induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- FPM flat pattern membrane
- Figure 2b is through the SEM and AFM of a flat pattern membrane (FPM) of a flat pattern prepared as a comparative example to confirm the effect of the nanostructure of the induced pluripotent stem cell culture plate according to an embodiment of the present invention The photographing results are shown.
- FPM flat pattern membrane
- Figure 3a is a schematic diagram of the production of an irregular pattern membrane (Irregular pattern Membrane; IPM) for comparing the effect of the nanostructures of induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- IPM irregular pattern Membrane
- Figure 3b is taken by SEM and AFM of Irregular pattern membrane (Irregular pattern Membrane; IPM) produced as a comparative example to confirm the effect of the nanostructure of the induced pluripotent stem cell culture plate according to an embodiment of the present invention The results are shown.
- IPM Irregular pattern Membrane
- Figure 3c is a schematic diagram of an irregular pattern membrane (Irregular pattern Membrane; IPM) prepared as a comparative example to confirm the effect of the nanostructure of the induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- IPM irregular pattern Membrane
- Figure 4 is a hemispherical, curved arc and Groove and cylindrical pattern (Groove pattern Membrane; GPM and Post pattern Membrane; PPM) in induced pluripotent stem cell culture plate according to an embodiment of the present invention Production schematic.
- Figure 5a is a schematic and SEM image of the Groove master mold in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- Figure 5b is a schematic diagram and SEM image of the post master mold in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- Figure 5c is a schematic diagram of SEM, AFM image and size of GPM in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- Figure 5d is a schematic diagram of SEM, AFM image and size of PPM in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- Figure 6a shows a comparison of the contact angle and the contact angle values before and after fibronectin coating on the culture membrane surface in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- Figure 6b shows an SEM image after fibronectin coating on each culture membrane surface in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- FIG. 7 is a phase photograph (A), Total PDL and PDL (B), and Ki67 expression in subcultures by attaching induced pluripotent stem cells to respective culture membranes in induced pluripotent stem cell culture plates according to an embodiment of the present invention. And immunochemical staining (C) results.
- Figure 8 is Oct3 / 4 and Nanog expression amount (A), immunochemical staining (B) and% expression cell volume (C) to confirm the undifferentiated state maintenance ability in induced pluripotent stem cell culture plate according to an embodiment of the present invention ).
- Figure 9 shows the SEM (A) and AFM (B) images of the morphological appearance of the cells during the cell attachment of induced pluripotent stem cells by induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- Figure 10 shows the change in the signal transduction system of the induced pluripotent stem cells by induced pluripotent stem cell culture plate according to an embodiment of the present invention through real-time PCR (A) and western blot (B) results.
- Figure 11 shows the change in the cytoskeleton of induced pluripotent stem cells by the induced pluripotent stem cell culture plate according to an embodiment of the present invention.
- induced pluripotent stem cell culture plate may comprise a culture membrane and a modifier.
- Induced pluripotent stem cell culture plate through the patterned culture membrane formed through the convex and recessed portion of the nano unit and the modifying agent coated on the surface of the culture membrane, to the growth of the induced pluripotent stem cells May affect
- the surface-modified culture plate reproduced the effects of extracellular matrix by using nanotopography.
- stem cells were maintained, and a large amount of induced pluripotent stem cells having excellent proliferation and adhesion ability were maintained. It is expected to be obtained as.
- the culture membrane is a configuration that provides a space in which the cells are cultured, and as shown in FIG. 1, a plurality of convex portions and concave portions are arranged so that the influence of extracellular matrix is patterned using nanotopography. Can be provided.
- the culture membrane may be preferably formed on a substrate.
- the culture membrane may preferably be made of polydimethylsiloxane (PDMS), but is not limited thereto.
- the iron portion preferably has a diameter of 300 to 400 nm, and more preferably 300 nm, but is not limited thereto.
- the recessed portion preferably has a diameter of 100 to 300 nm, but is not limited thereto. It doesn't happen.
- the convex portion and the recess portion may be preferably formed in the shape of hemispherical, curved arc shape, corrugated shape or column shape, but is not limited thereto.
- the induced pluripotent stem cell culture plate according to an embodiment of the present invention was coated with a modifier on the surface of the culture membrane, thereby reducing the contact angle of the culture membrane, thereby improving the hydrophilicity of stem cells. Can enhance the adhesion.
- the modifier may be coated (blue) on the culture membrane surface, as shown in FIG. 1, thereby increasing hydrophilization.
- the modifier may preferably be fibronectin, but is not limited thereto.
- induced pluripotent stem cell culture plates having various patterns were prepared (see Examples 1 and 2), and the results of culturing induced pluripotent stem cells using the prepared culture plates were as follows. As the passage progressed compared to the culture, it was confirmed that the induced pluripotent stem cells filling the culture membrane tightly and improved the proliferation capacity. In addition, it was confirmed that the undifferentiated maintenance ability of stem cells was improved by confirming the expression level of undifferentiated embryonic stem cell markers (see Example 3).
- the induced pluripotent stem cell culture plate and / or the proliferation of induced pluripotent stem cells in accordance with an embodiment of the present invention while maintaining the stem cell characteristics of the induced pluripotent stem cells to be able to culture and / or obtain It is expected.
- the present invention provides a method for culturing induced pluripotent stem cells using an induced pluripotent stem cell culture plate comprising the following steps:
- step (d) culturing by attaching induced pluripotent stem cells to the culture table prepared in step (c).
- step (a) a plurality of convex portions and concave portions are arranged on the upper surface of the substrate to form a culture membrane.
- the substrate may be formed of a silicon wafer or a glass wafer, but is not limited thereto.
- etching is performed using the Deep RIE equipment.
- silane treatment polydimethylsiloxane (PDMS) is applied and placed in a vacuum desiccator for 3 to 5 hours, and then the substrate is heated at 70 to 90 ° C. for 11 to 13 hours.
- a culture membrane in which a plurality of recesses are patterned can be disposed on the substrate.
- step (b) the patterned culture membrane obtained in step (a) is hydrophilized. More specifically, the patterned culture membrane obtained in step (a) using the PDMS shows a hydrophobicity due to the characteristics of the PDMS, it is necessary to hydrophilize it, it is preferable that the oxygen plasma treatment for this. At this time, the oxygen plasma treatment is preferably carried out under an oxygen amount of 25 SCCM, a power of 40 W, and 40 SEC.
- step (c) the hydrophilized culture membrane obtained in step (b) is coated with fibronectin.
- the fibronectin is not directly coated on the culture membrane, it is pretreated with poly-DL-ornithine for 4 hours or more, and then coated with fibronectin for 1 hour or more.
- the induced pluripotent stem cell culture plate according to the embodiment of the present invention can be obtained.
- step (d) induced pluripotent stem cells are attached to the induced pluripotent stem cell culture plate obtained in step (c), and passaging is performed.
- a flat pattern pattern membrane which is a culture membrane without a pattern is formed as a comparison group .
- PDMS polydimethylsiloxane
- SEM scanning electron microscopy
- AFM atomic force microscopy
- Example 1-1 in order to confirm the effect of the nano-topography according to the pattern of the induced pluripotent stem cell culture plate according to the present invention, as shown in Figure 3a, an irregular pattern membrane (IPM) was prepared.
- IPM irregular pattern membrane
- the primer on the glass wafer (quartz)
- it is dried for 2 hours.
- the nanoprint was sprayed onto the dried glass wafer, dried at room temperature, and then PDMS was applied to the dried glass wafer, and then placed in a vacuum desiccator for 2 hours.
- the glass wafer was heated to 80 ° C. for 12 hours to obtain an irregular culture membrane (IPM), which was confirmed through SEM and AFM images.
- IPM irregular culture membrane
- FIG. 3B the irregular surface was confirmed.
- FIG. 3C it was confirmed that an irregular pattern membrane having an average surface height of 87.1 nm and a distance between particle patterns was 226.2 to 447.6 nm.
- a photoresist pattern is formed on a silicon wafer using an E-beam lithography apparatus, and then the patterned silicon is formed.
- the wafer was etched using a Deep RIE (reactive ion etcher) device.
- the surface of the patterned silicon wafer was converted to hydrophobicity by silane treatment, and then PDMS was poured on it and placed in a vacuum desiccator for 4 hours. Thereafter, the silicon wafer was heated at a temperature of 80 ° C. for 12 hours, and as shown in FIG. 4, a Groove pattern membrane (GPM) and a Post pattern membrane (PPM) were obtained.
- GPM Groove pattern membrane
- PPM Post pattern membrane
- the GPM master mold had a width of 239.7 nm and a height of 98.5 nm. Groove-shaped master molds with a spacing of 237.9 nm were identified, and in the case of PPM, 237.2 nm in width, 98.5 nm in height, and 237.2 nm post-shaped master mold were found.
- the intaglio and embossed directions of the master mold were respectively reversed, and the GPM and the PPM were confirmed. More specifically, in the case of GPM, the width was 377.2 nm, the height was 82.5 nm, and the interval was 161.1 nm. The GPM was confirmed. In the case of PPM, the width was 310 nm, the height was 60.1 nm, and the interval was 100.7 nm.
- the culture membranes prepared in Example 1 were all made of PDMS to have hydrophobic, and were subjected to hydrophilization treatment to modify the hydrophilic surface through oxygen plasma treatment. More specifically, the oxygen plasma treatment was performed under conditions of an oxygen content of 25 SCCM, a power of 40 W, and a time of 40 SEC.
- the modifier according to the embodiment of the present invention was intended to increase the hydrophilization of the culture membrane. . More specifically, the surface of each culture membrane subjected to oxygen plasma treatment was coated with a modifier and the contact angle was measured. At this time, fibronectin was used as a modifier.
- Example 3 According to the present invention Induced pluripotent stem cells Of culture plate Induced pluripotent stem cells Culture capacity check
- induced pluripotent stem cells were passaged every 5 days and observed changes. Proliferation patterns of induced pluripotent stem cells were observed at passage 1 (passage 1; P 1) and passage 5 (P 5).
- induced pluripotent stem cells on GPM and PPM were aggregated in the form of colonies in P 1 and grew and did not proliferate properly, but did not form colonies as passage was progressed. As a result, the cells were grown normally and filled with culture membranes.
- Factors related to cell adhesion using induced pluripotent stem cells cultured on induced pluripotent stem cell culture plates according to the present invention such as Focal adhesion kinase, which transmits and amplifies external signals initiated through integrin receptors (FAK), a-actinin, Paxillin, Vinculin, Talin and Zyxin expression levels of proteins were confirmed by real-time PCR and western blot.
- FAK integrin receptors
- a-actinin, Vinculin and Paxillin increased the overall expression as passage in the culture membrane, FAK was faster than other factors only in GPM and PPM P 6
- Talin and Zyxin showed different expressions from other adhesion factors.
- the protein expression level of p-FAK was found to be slightly higher in P 6 and P 10.
- the phosphorylation of Paxillin which is known to regulate the major physiological phenomena of cells by binding to FAK, was also cultured. The initial culture at was high in the FPM, but as the passage progressed it was confirmed that the higher in the GPM and PPM.
- the cytoskeleton plays a very important role in the final step of recognizing the difference in force on the physical stimulation through extracellular matrix and determining the behavior of the cell.
- the change in the cytoskeleton was confirmed by Phalloidin staining.
- Induced pluripotent stem cell culture plate is a patterned culture membrane and a modifier coated on the surface of the culture membrane to reproduce the effects of physical microenvironment, that is, extracellular matrix in vivo using nanotopography
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Abstract
The present invention relates to an induced pluripotent stem cell culture plate and, more particularly, to an induced pluripotent stem cell culture plate which enables acquisition of a large amount of induced pluripotent stem cells, having maintained stemness and excellent proliferation and adhesion properties, by means of a culture membrane, which is a reproduction of the influence of the extracellular matrix by means of nanotopography and has a plurality of prominent parts and depressed parts aligned and forming a pattern, and a modifier which coats the surface of the culture membrane and thus enhances the adhesion properties.
Description
본 발명은 유도만능줄기세포 배양 플레이트에 관한 것으로서, 보다 구체적으로는, 상기 배양 플레이트 표면을 나노 단위로 패턴화하여 세포외기질의 영향을 재현하고, 피브로넥틴으로 코팅시켜 배양 플레이트와 세포의 접촉각을 감소시킴으로서, 줄기세포성이 유지되면서, 증식 및 부착 능력이 우수한 유도만능줄기세포를 다량으로 획득이 가능한 유도만능줄기세포 배양 플레이트에 관한 것이다.The present invention relates to an induced pluripotent stem cell culture plate, and more specifically, by reproducing the effect of extracellular matrix by patterning the surface of the culture plate in nano units, and coating with fibronectin to reduce the contact angle between the culture plate and the cells. In addition, while maintaining the stem cell properties, it relates to an induced pluripotent stem cell culture plate capable of obtaining a large amount of induced pluripotent stem cells excellent in proliferation and adhesion ability.
줄기세포는 증식 능력 및 다양한 종류의 세포로 분화할 수 있는 독특한 능력을 가지며, 조직공학 및 재생의약에 있어서 많은 대안과 기회를 제공하고 있다. 줄기세포는 체내에서 용해성 및 비용해성 인자의 복합체 및 조절된 생화학적 화합물과 같이 유용하고 조직 특이적인 환경 하에 존재한다.Stem cells have a proliferative capacity and unique ability to differentiate into various cell types, and provide many alternatives and opportunities in tissue engineering and regenerative medicine. Stem cells exist in useful and tissue specific environments such as complexes of soluble and insoluble factors and regulated biochemical compounds in the body.
최근, 줄기세포를 체외에서 배양한 후, 이를 이용하는 세포 치료가 점차 확대되고 있다. 이에, 세포의 증식과 분화 효율을 향상시킬 수 있는 배양 방법과 배양계(culture system)에 대한 관심이 높아지고 있다. 특히, 배양계에는 다양한 기기들이 관계하고 있으며, 이에 있어서 중요한 요소 중의 하나가 세포 배양용 용기이다. 일반적으로 많은 수의 세포를 얻기 위하여, 배양 세포의 특성에 따라 인공적으로 만들어진 배양 접시, 배양 플라스크 또는 롤러 병 등의 세포 배양용 용기에서 세포를 배양하게 된다. 이 때, 인공적으로 배양하는 세포들은 주로 세포 배양용 용기의 바닥에 부착되어 성장, 증식 및 분화의 과정을 거치면서 생존하지만, 일부 세포는 여러 층을 형성하면서, 다른 세포 위에 겹쳐 증식하기도 하고, 또 다른 일부 세포는 세포 배양액 내에서 부유 상태를 유지하면서 성장 증식 및 분화하기도 한다. 이로 인해, 인공적으로 만들어진 세포 배양용 용기는 원래 세포가 안주하고 있는 세포외 기질과는 다른 표면 특성을 가지고 있는바, 세포 증식 및 분화 효율이 저하될 수 있다.In recent years, after culturing stem cells in vitro, cell treatment using the same has been gradually expanded. Thus, there is a growing interest in culture methods and culture systems that can improve the proliferation and differentiation efficiency of cells. In particular, various devices are involved in the culture system, and one of the important factors is a cell culture container. In general, in order to obtain a large number of cells, cells are cultured in a cell culture vessel such as a culture dish, a culture flask, or a roller bottle artificially made according to the characteristics of the cultured cells. At this time, the artificially cultured cells are mainly attached to the bottom of the cell culture vessel to survive the process of growth, proliferation and differentiation, but some cells multiply and multiply on other cells, Some other cells may grow, grow, and differentiate while remaining suspended in cell culture. As a result, the artificially made cell culture vessel has a surface property different from that of the extracellular matrix in which the cells are originally placed, and thus, cell proliferation and differentiation efficiency may be reduced.
한편, 나노 미터 크기의 정밀성을 가지는 미세 패턴 제작 기술은 최근 반도체 공업의 발전에 크게 힘입은 바 있으며, 이를 이용한 온전자선 기술, 주사탐침 현미경 기술, 나노 임프린트 기술, 마이크로 컨택 프린트 기술 등 다양한 나노 미터 크기의 제어기술 들과 함께 이를 이용한 응용기술의 개발을 가능하게 하고 있다. 이로 인해, 패턴을 주형으로 음각의 복제 패턴을 만들어 내는데도 이용할 수 있으며, 이 기술은 가공성 및 표면의 화학적 개질 등이 용이한 고분자 기판 위에 쉽고 빠르게 패턴을 전사할 수 있다.On the other hand, micro pattern fabrication technology with nanometer precision has been greatly helped by the development of the semiconductor industry in recent years, and various nanometer sizes including on-electron beam technology, scanning probe microscope technology, nano imprint technology, and micro contact printing technology Along with the control technologies of, it is possible to develop application technology using them. As a result, the pattern can be used as a mold to create an intaglio copy pattern, and this technology can transfer a pattern quickly and easily onto a polymer substrate which is easy to process and chemically modify the surface.
또한, 세포 거동을 제어하기 위한 연구는 다양하고도 폭 넓은 의학적 응용 가능성 때문에 여러 가지 기술들이 개발되고 있는 가운데, 특히, 줄기세포의 분화를 제어하는 것은 그 임팩트가 매우 클 것으로 판단되어, 최근 주목 받고 있다. 종래 줄기세포의 분화를 비롯한 세포의 여러 가지 거동 제어는 세포에 해당 신호를 전달하는 화학 물질들을 이용하는 것이 일반적이었으나, 상술한 미세 패턴을 이용하여 세포의 거동을 제어하는 연구가 진행되고 있으며, 미세 패턴의 크기 또한 종래의 마이크로 단위에서 나노 패턴으로 발전하고 있다.In addition, researches for controlling cell behavior have been developed due to various and wide range of medical applications, and in particular, controlling the differentiation of stem cells is considered to have a great impact. have. Conventionally, the control of various behaviors of cells, including the differentiation of stem cells, has generally been accomplished by using chemicals that transmit a signal to cells. However, studies to control the behavior of cells using the aforementioned fine patterns have been conducted. The size of is also evolving into nano-patterns in conventional micro-units.
이러한 나노 단위의 패턴을 이용하여 특정 세포의 배양능력을 향상시키거나, 줄기세포성을 유지하면서, 증식 능력 및 분화 능력을 향상시키는 배양용 용기 개발이 주요한 과제의 대상이 되고 있고, 이에 대한 연구가 이루어지고 있으나(한국공개특허 10-2010-0039944), 아직은 미비한 실정이다.The development of a container for cultivation to improve the cultivation capacity of a specific cell by using the nano-unit pattern or to improve the proliferative capacity and differentiation capacity while maintaining stem cell nature has been the subject of a major problem. Although it is made (Korean Patent Publication No. 10-2010-0039944), it is still inadequate.
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로서, 본 발명자들은 나노 단위의 철부 및 요부를 구비하는 배양 멤브레인 및 상기 멤브레인 표면에 개질제를 이용한 코팅을 통해서, 줄기세포성을 유지하면서 증식 및 부착 능력이 우수한 유도만능줄기세포를 배양하는 배양 플레이트를 발명하였다.The present invention has been made to solve the above problems, the present inventors are proliferating and attaching while maintaining stem cellity through a culture membrane and a coating using a modifier on the surface of the membrane and the nano-structure iron and recesses Invented a culture plate for culturing induced pluripotent stem cells with excellent ability.
이에, 본 발명의 목적은Thus, the object of the present invention
300-400㎚의 직경을 갖는 철부 및 100-300㎚의 직경을 갖는 요부가 복수개로 배열되어 패턴을 형성하는 배양 멤브레인; 및A culture membrane in which a convex portion having a diameter of 300-400 nm and a concave portion having a diameter of 100-300 nm are arranged in a plurality to form a pattern; And
상기 배양 멤브레인 표면에 코팅된 개질제를 포함하는, 유도만능줄기세포 배양 플레이트를 제공하는 것이다.To provide an induced pluripotent stem cell culture plate comprising a modifier coated on the culture membrane surface.
본 발명의 다른 목적은Another object of the present invention
하기의 단계를 포함하는 유도만능줄기세포 배양 플레이트를 이용한 유도만능줄기세포 배양 방법:Induced pluripotent stem cell culture method using induced pluripotent stem cell culture plate comprising the following steps:
(a) 300-400㎚의 직경을 갖는 복수개의 철부 및 100-300㎚의 직경을 갖는 요부가 복수개로 배열되어 패턴을 형성하는 배양 멤브레인을 기판 상면에 배치시키는 단계;(a) disposing a plurality of convex portions having a diameter of 300-400 nm and a plurality of concave portions having a diameter of 100-300 nm arranged on a top surface of the substrate to form a pattern;
(b) 상기 배양 멤브레인 표면을 친수성으로 개질시키는 단계;(b) modifying the culture membrane surface to be hydrophilic;
(c) 개질된 상기 배양 멤브레인 표면을 개질제로 코팅시키는 단계; 및(c) coating the modified culture membrane surface with a modifier; And
(d) 상기 (c) 단계에서 제조된 배양 테이블에 유도만능줄기세포를 부착하여 배양하는 단계를 제공하는 것이다.(d) attaching the induced pluripotent stem cells to the culture table prepared in step (c) to provide a step of culturing.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기 목적을 달성하기 위하여, 본 발명은 In order to achieve the above object, the present invention
300-400㎚의 직경을 갖는 철부 및 100-300㎚의 직경을 갖는 요부가 복수개로 배열되어 패턴을 형성하는 배양 멤브레인; 및A culture membrane in which a convex portion having a diameter of 300-400 nm and a concave portion having a diameter of 100-300 nm are arranged in a plurality to form a pattern; And
상기 배양 멤브레인 표면에 코팅된 개질제를 포함하는, 유도만능줄기세포 배양 플레이트를 제공한다.It provides an induced pluripotent stem cell culture plate comprising a modifier coated on the culture membrane surface.
바람직하게는, 상기 배양 멤브레인은, 폴리디메틸실록산(PDMS)으로 이루어질 수 있다.Preferably, the culture membrane may be made of polydimethylsiloxane (PDMS).
바람직하게는, 상기 개질제는 피브로넥틴 (Fibronectin)일 수 있다.Preferably, the modifier may be Fibronectin.
바람직하게는, 상기 철부 및 요부는, 반구형, 만곡진 호형, 파형 또는 기둥형으로 이루어질 수 있다.Preferably, the convex portion and the recessed portion may be formed in a hemispherical shape, curved arc shape, wave shape or column shape.
바람직하게는, 상기 배양 플레이트는, 유도만능줄기세포 (Induced pluripotent stem cells)의 부착 및 증식능을 향상시킬 수 있다.Preferably, the culture plate, it is possible to improve the adhesion and proliferation of induced pluripotent stem cells (Induced pluripotent stem cells).
바람직하게는, 상기 배양 플레이트는, 유도만능줄기세포 (Induced pluripotent stem cells)의 미분화 유지능을 향상시킬 수 있다.Preferably, the culture plate, it is possible to improve the undifferentiated maintenance of induced pluripotent stem cells (Induced pluripotent stem cells).
본 발명은The present invention
하기의 단계를 포함하는 유도만능줄기세포 배양 플레이트를 이용한 유도만능줄기세포 배양 방법:Induced pluripotent stem cell culture method using induced pluripotent stem cell culture plate comprising the following steps:
(a) 300-400㎚의 직경을 갖는 복수개의 철부 및 100-300㎚의 직경을 갖는 요부가 복수개로 배열되어 패턴을 형성하는 배양 멤브레인을 기판 상면에 배치시키는 단계;(a) disposing a plurality of convex portions having a diameter of 300-400 nm and a plurality of concave portions having a diameter of 100-300 nm arranged on a top surface of the substrate to form a pattern;
(b) 상기 배양 멤브레인 표면을 친수성으로 개질시키는 단계;(b) modifying the culture membrane surface to be hydrophilic;
(c) 개질된 상기 배양 멤브레인 표면을 개질제로 코팅시키는 단계; 및(c) coating the modified culture membrane surface with a modifier; And
(d) 상기 (c) 단계에서 제조된 배양 테이블에 유도만능줄기세포를 부착하여 배양하는 단계를 제공한다.(d) providing a step of culturing by attaching induced pluripotent stem cells to the culture table prepared in step (c).
본 발명에 따른 유도만능줄기세포 배양 플레이트는, 생체내에서의 물리적 미세환경 즉, 세포외기질의 영향을 나노지형 (nanotopography)을 이용하여 재현하는 패턴화된 배양 멤브레인 및 상기 배양 멤브레인 표면에 코팅된 개질제를 포함함으로서, 상기 배양 플레이트에 사람유도만능줄기세포를 부착하여 배양하였을 때, 종래의 줄기세포의 계대배양 과정 중에서 손실되는 줄기세포성 유지의 문제를 해결하였는바, 증식 능력 및 부착 능력이 보다 우수하고, 미분화 유지능이 향상된 사람유도만능줄기세포를 다량 획득할 수 있다. Induced pluripotent stem cell culture plate according to the present invention is a patterned culture membrane and a modifier coated on the surface of the culture membrane to reproduce the effects of physical microenvironment, that is, extracellular matrix in vivo using nanotopography By including, when the human induced pluripotent stem cells attached to the culture plate and cultured, the problem of stem cell maintenance that is lost during the passage of the conventional stem cells, bar proliferation ability and adhesion ability is more excellent In addition, a large amount of human induced pluripotent stem cells having improved undifferentiated maintenance ability can be obtained.
도 1은 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트의 분해 사시도이다.1 is an exploded perspective view of an induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 2a는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트의 나노지형 (nanotopography)에 따른 영향을 비교하기 위한 편평한 형태의 패턴 멤브레인 (Flat pattern Membrane; FPM)의 제작 모식도를 나타낸 것이다.Figure 2a shows a schematic diagram of a flat pattern membrane (FPM) of the flat form for comparing the effect of the nanotopography (nanotopography) of the induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 2b는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트의 나노지형에 따른 영향을 확인하기 위한 비교예로 제작된 편평한 형태의 패턴 멤브레인 (Flat pattern Membrane; FPM)의 SEM 및 AFM를 통해 촬영한 결과를 나타낸 것이다.Figure 2b is through the SEM and AFM of a flat pattern membrane (FPM) of a flat pattern prepared as a comparative example to confirm the effect of the nanostructure of the induced pluripotent stem cell culture plate according to an embodiment of the present invention The photographing results are shown.
도 3a는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트의 나노지형에 따른 영향을 비교하기 위한 불규칙한 패턴 멤브레인 (Irregular pattern Membrane; IPM)의 제작 모식도이다.Figure 3a is a schematic diagram of the production of an irregular pattern membrane (Irregular pattern Membrane; IPM) for comparing the effect of the nanostructures of induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 3b는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트의 나노지형에 따른 영향을 확인하기 위한 비교예로 제작된 불규칙한 패턴 멤브레인 (Irregular pattern Membrane; IPM)의 SEM 및 AFM를 통해 촬영한 결과를 나타낸 것이다.Figure 3b is taken by SEM and AFM of Irregular pattern membrane (Irregular pattern Membrane; IPM) produced as a comparative example to confirm the effect of the nanostructure of the induced pluripotent stem cell culture plate according to an embodiment of the present invention The results are shown.
도 3c는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트의 나노지형에 따른 영향을 확인하기 위한 비교예로 제작된 불규칙한 패턴 멤브레인 (Irregular pattern Membrane; IPM)의 모식도이다.Figure 3c is a schematic diagram of an irregular pattern membrane (Irregular pattern Membrane; IPM) prepared as a comparative example to confirm the effect of the nanostructure of the induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 4는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에서 반구형, 만곡진 호형 및 파형(Groove) 및 원기둥형(Post) 패턴 멤브레인 (Groove pattern Membrane; GPM 및 Post pattern Membrane; PPM)의 제작 모식도이다.Figure 4 is a hemispherical, curved arc and Groove and cylindrical pattern (Groove pattern Membrane; GPM and Post pattern Membrane; PPM) in induced pluripotent stem cell culture plate according to an embodiment of the present invention Production schematic.
도 5a는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에서 Groove master mold의 모식도 및 SEM 이미지이다.Figure 5a is a schematic and SEM image of the Groove master mold in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 5b는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에서 Post master mold의 모식도 및 SEM 이미지이다.Figure 5b is a schematic diagram and SEM image of the post master mold in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 5c는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에서 GPM의 SEM, AFM 이미지 및 크기 모식도이다.Figure 5c is a schematic diagram of SEM, AFM image and size of GPM in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 5d는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에서 PPM의 SEM, AFM 이미지 및 크기 모식도이다.Figure 5d is a schematic diagram of SEM, AFM image and size of PPM in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 6a는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에서 배양 멤브레인 표면상에 피브로넥틴 코팅 전/후의 접촉각 및 접촉각 수치의 비교를 나타낸 것이다.Figure 6a shows a comparison of the contact angle and the contact angle values before and after fibronectin coating on the culture membrane surface in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 6b는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에서 각 배양 멤브레인 표면상에 피브로넥틴 코팅 후의 SEM 이미지를 나타낸 것이다.Figure 6b shows an SEM image after fibronectin coating on each culture membrane surface in induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 7은 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에서 각 배양 멤브레인에 유도만능줄기세포를 부착하여 계대 배양에서 Phase 사진 (A), Total PDL 및 PDL (B), 및 Ki67 발현량 및 면역화학염색 (C) 결과를 나타낸 것이다.7 is a phase photograph (A), Total PDL and PDL (B), and Ki67 expression in subcultures by attaching induced pluripotent stem cells to respective culture membranes in induced pluripotent stem cell culture plates according to an embodiment of the present invention. And immunochemical staining (C) results.
도 8은 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에서 미분화상태 유지능을 확인하기 위한 Oct3/4 및 Nanog 발현량 (A), 면역화학염색 (B) 및 발현 세포량 % (C)를 나타낸 것이다.Figure 8 is Oct3 / 4 and Nanog expression amount (A), immunochemical staining (B) and% expression cell volume (C) to confirm the undifferentiated state maintenance ability in induced pluripotent stem cell culture plate according to an embodiment of the present invention ).
도 9는 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에 의한 유도만능줄기세포의 세포 부착시 세포의 형태학적 모양을 SEM (A) 및 AFM (B) 이미지를 나타낸 것이다.Figure 9 shows the SEM (A) and AFM (B) images of the morphological appearance of the cells during the cell attachment of induced pluripotent stem cells by induced pluripotent stem cell culture plate according to an embodiment of the present invention.
도 10은 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에 의한 유도만능줄기세포의 부착 관계 신호전달체계 변화를 real-time PCR (A) 및 western blot (B) 결과를 통해 나타낸 것이다.Figure 10 shows the change in the signal transduction system of the induced pluripotent stem cells by induced pluripotent stem cell culture plate according to an embodiment of the present invention through real-time PCR (A) and western blot (B) results.
도 11은 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트에 의한 유도만능줄기세포의 세포골격의 변화를 나타낸 것이다.Figure 11 shows the change in the cytoskeleton of induced pluripotent stem cells by the induced pluripotent stem cell culture plate according to an embodiment of the present invention.
이하, 첨부된 도면을 참조하여 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 본 발명을 용이하게 실시할 수 있도록 바람직한 실시예를 상세히 설명한다. 본 발명의 실시예를 설명하기 위한 도면에 개시된 형상, 크기, 비율, 각도, 개수 등은 예시적인 것이므로 본 발명이 도시된 사항에 한정되는 것은 아니다. 다만, 본 발명의 바람직한 실시예를 상세하게 설명함에 있어, 관련된 공지 기능 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략한다. 또한, 유사한 기능 및 작용을 하는 부분에 대해서는 도면 전체에 걸쳐 동일한 부호를 사용한다.Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present invention. Shapes, sizes, ratios, angles, numbers, and the like disclosed in the drawings for describing the embodiments of the present invention are exemplary, and the present invention is not limited to the illustrated items. However, in describing the preferred embodiment of the present invention in detail, if it is determined that the detailed description of the related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. In addition, the same reference numerals are used throughout the drawings for parts having similar functions and functions.
덧붙여, 명세서 전체에서, 어떤 부분이 다른 부분과 '연결' 되어 있다고 할 때, 이는 '직접적으로 연결' 되어 있는 경우뿐만 아니라, 그 중간에 다른 소자를 사이에 두고 '간접적으로 연결' 되어 있는 경우도 포함한다. 또한, 어떤 구성요소를 '포함' 한다는 것은, 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있다는 것을 의미한다.In addition, in the specification, when a part is 'connected' to another part, it is not only 'directly connected' but also 'indirectly connected' with another element in between. Include. In addition, the term 'comprising' of an element means that the element may further include other elements, not to exclude other elements unless specifically stated otherwise.
도 1은 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트의 사시도를 도시한 것이다. 도 1에 도시된 바와 같이, 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트는 배양 멤브레인 및 개질제를 포함하여 구성될 수 있다.1 is a perspective view of an induced pluripotent stem cell culture plate according to an embodiment of the present invention. As shown in Figure 1, induced pluripotent stem cell culture plate according to an embodiment of the present invention may comprise a culture membrane and a modifier.
본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트는, 나노 단위의 철부 및 요부를 통하여 형성된 패턴화된 배양 멤브레인 및 상기 배양 멤브레인 표면에 코팅된 개질제를 통해서, 유도만능줄기세포의 배양 증식에 영향을 줄 수 있다. 이와 같은 구성을 채택함으로써, 표면이 개질된 배양 플레이트는 세포외기질의 영향을 나노지형 (nanotopography)을 이용하여 재현하였는바, 줄기세포성이 유지되면서, 증식 및 부착 능력이 우수한 유도만능줄기세포를 다량으로 획득할 수 있을 것으로 기대된다.Induced pluripotent stem cell culture plate according to an embodiment of the present invention, through the patterned culture membrane formed through the convex and recessed portion of the nano unit and the modifying agent coated on the surface of the culture membrane, to the growth of the induced pluripotent stem cells May affect By adopting such a configuration, the surface-modified culture plate reproduced the effects of extracellular matrix by using nanotopography. As a result, stem cells were maintained, and a large amount of induced pluripotent stem cells having excellent proliferation and adhesion ability were maintained. It is expected to be obtained as.
이하에서는, 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트를 구성하는 각각의 구성요소에 대하여 상세히 설명하기로 한다.Hereinafter, each component constituting the induced pluripotent stem cell culture plate according to an embodiment of the present invention will be described in detail.
배양 멤브레인은, 세포의 배양이 이루어지는 공간을 제공하는 구성으로서, 도 1에 도시된 바와 같이, 철부 및 요부가 복수개로 배열되어 세포외기질의 영향을 나노지형 (nanotopography)을 이용하여 패턴화한 물리적 환경을 제공할 수 있다. 이 때, 상기 배양 멤브레인은 바람직하게는, 기판 상에 형성될 수 있다. 또한, 상기 배양 멤브레인은 바람직하게는, 폴리디메틸실록산(PDMS)으로 이루어질 수 있으나, 이에 제한되는 것은 아니다. 더욱이, 상기 철부는 300 ~ 400 ㎚의 직경을 갖는 것이 바람직하며, 더욱 바람직하게는 300 ㎚일 수 있으나, 이에 제한되는 것은 아니며, 상기 요부는 100 ~ 300 ㎚의 직경을 갖는 것이 바람직하나, 이에 제한되는 것은 아니다. 더욱이, 상기 철부 및 요부는 바람직하게는 반구형, 만곡진 호형, 파형 또는 기둥형의 형태로 이루어질 수 있으나, 이에 제한되는 것은 아니다.The culture membrane is a configuration that provides a space in which the cells are cultured, and as shown in FIG. 1, a plurality of convex portions and concave portions are arranged so that the influence of extracellular matrix is patterned using nanotopography. Can be provided. At this time, the culture membrane may be preferably formed on a substrate. In addition, the culture membrane may preferably be made of polydimethylsiloxane (PDMS), but is not limited thereto. Further, the iron portion preferably has a diameter of 300 to 400 nm, and more preferably 300 nm, but is not limited thereto. The recessed portion preferably has a diameter of 100 to 300 nm, but is not limited thereto. It doesn't happen. Further, the convex portion and the recess portion may be preferably formed in the shape of hemispherical, curved arc shape, corrugated shape or column shape, but is not limited thereto.
한편, 줄기세포를 배양하는데 있어서, 종래의 배양기들은 산소 플라즈마를 패턴화된 배양기 상에 처리하여 줄기세포의 부착능을 강화시켜왔다. 하지만, 산소 플라즈마 처리만으로는 친수화가 충분히 이루어지지 않는바, 상기 줄기세포의 만족스러운 부착능을 얻지 못하는 실정이었다. 이러한 문제점을 해결하기 위해 본 발명의 일실시예에 따른 유도만능줄기세포 배양 플레이트는 상기 배양 멤브레인 표면을 개질제로 코팅하였는바, 이를 통해 상기 배양 멤브레인의 접촉각을 감소시켜, 친수화가 향상됨에 따라 줄기세포의 부착능을 강화시킬 수 있다.On the other hand, in culturing stem cells, conventional incubators have enhanced the adhesion of stem cells by treating oxygen plasma on a patterned incubator. However, since only the oxygen plasma treatment does not sufficiently hydrophilize, it was not possible to obtain satisfactory adhesion of the stem cells. In order to solve this problem, the induced pluripotent stem cell culture plate according to an embodiment of the present invention was coated with a modifier on the surface of the culture membrane, thereby reducing the contact angle of the culture membrane, thereby improving the hydrophilicity of stem cells. Can enhance the adhesion.
보다 구체적으로, 개질제는, 도 1에 도시된 바와 같이, 상기 배양 멤브레인 표면에 코팅될 수 있으며(파란색), 이로 인해 친수화를 증가시킬 수 있다. 이 때, 상기 개질제은 바람직하게는, 피브로넥틴 (Fibronectin)일 수 있으나, 이에 제한되는 것은 아니다.More specifically, the modifier may be coated (blue) on the culture membrane surface, as shown in FIG. 1, thereby increasing hydrophilization. At this time, the modifier may preferably be fibronectin, but is not limited thereto.
본 발명의 일 실시예에서는, 다양한 패턴을 갖는 유도만능줄기세포 배양 플레이트를 제작하고(실시예 1 및 2 참조), 상기 제작된 배양 플레이트를 이용하여 유도만능줄기세포의 배양 능력을 확인한 결과, 초기 배양에 비하여 계대가 진행됨에 따라 배양 멤브레인 상을 꽉 채우는 유도만능줄기세포의 부착 및 증식능 향상을 확인하였다. 뿐만 아니라, 미분화 배아줄기세포 마커의 발현량 확인을 통해서, 줄기세포의 미분화 유지능도 향상됨을 확인하였다(실시예 3 참조).In one embodiment of the present invention, induced pluripotent stem cell culture plates having various patterns were prepared (see Examples 1 and 2), and the results of culturing induced pluripotent stem cells using the prepared culture plates were as follows. As the passage progressed compared to the culture, it was confirmed that the induced pluripotent stem cells filling the culture membrane tightly and improved the proliferation capacity. In addition, it was confirmed that the undifferentiated maintenance ability of stem cells was improved by confirming the expression level of undifferentiated embryonic stem cell markers (see Example 3).
따라서, 본 발명에 일실시예에 따른 유도만능줄기세포 배양 플레이트를 통해서 유도만능줄기세포의 줄기세포성을 유지하면서, 세포 부착 및 증식이 우수한 유도만능줄기세포를 배양 및/또는 수득할 수 있을 것으로 기대된다.Therefore, the induced pluripotent stem cell culture plate and / or the proliferation of induced pluripotent stem cells in accordance with an embodiment of the present invention, while maintaining the stem cell characteristics of the induced pluripotent stem cells to be able to culture and / or obtain It is expected.
이에, 본 발명의 다른 양태로서, 본 발명은 하기의 단계를 포함하는 유도만능줄기세포 배양 플레이트를 이용한 유도만능줄기세포 배양 방법을 제공한다:Accordingly, as another aspect of the present invention, the present invention provides a method for culturing induced pluripotent stem cells using an induced pluripotent stem cell culture plate comprising the following steps:
(a) 300-400㎚의 직경을 갖는 복수개의 철부 및 100-300㎚의 직경을 갖는 요부가 복수개로 배열되어 패턴을 형성하는 배양 멤브레인을 기판 상면에 배치시키는 단계;(a) disposing a plurality of convex portions having a diameter of 300-400 nm and a plurality of concave portions having a diameter of 100-300 nm arranged on a top surface of the substrate to form a pattern;
(b) 상기 나노 배양 멤브레인 표면을 친수성으로 개질시키는 단계;(b) modifying the nanoculture membrane surface to be hydrophilic;
(c) 개질된 상기 배양 멤브레인 표면을 개질제로 코팅시키는 단계; 및(c) coating the modified culture membrane surface with a modifier; And
(d) 상기 (c) 단계에서 제조된 배양 테이블에 유도만능줄기세포를 부착하여 배양하는 단계.(d) culturing by attaching induced pluripotent stem cells to the culture table prepared in step (c).
단계 (a)에서는, 기판 상면에 철부 및 요부가 복수개로 배열되어 패턴을 형성하는 배양 멤브레인을 배치시킨다. 이 때, 상기 기판은, 실리콘 웨이퍼 또는 글라스 웨이퍼로 이루어질 수 있으나, 이에 제한되는 것은 아니다. In step (a), a plurality of convex portions and concave portions are arranged on the upper surface of the substrate to form a culture membrane. In this case, the substrate may be formed of a silicon wafer or a glass wafer, but is not limited thereto.
또한, 상기 기판 상에 배양 멤브레인을 배치시키기 위해서, E-beam 리소그래피 장비를 사용하여 기판 상에 포토 레지스트 패턴을 형성한 후, Deep RIE 장비를 이용하여 식각 (etching)을 진행 한다. 이 후, 실란 처리를 진행한 뒤, 폴리디메틸실록산 (PDMS)를 도포하여, 3 ~ 5 시간 동안 진공데시게이터에 놓은 후, 상기 기판을 70 ~ 90 ℃, 11 ~ 13 시간 가열을 진행하면, 철부 및 요부가 복수개로 패턴화된 배양 멤브레인을 기판 상에 배치할 수 있다.In addition, in order to arrange the culture membrane on the substrate, after forming a photoresist pattern on the substrate using an E-beam lithography equipment, etching is performed using the Deep RIE equipment. After the silane treatment, polydimethylsiloxane (PDMS) is applied and placed in a vacuum desiccator for 3 to 5 hours, and then the substrate is heated at 70 to 90 ° C. for 11 to 13 hours. And a culture membrane in which a plurality of recesses are patterned can be disposed on the substrate.
단계 (b)에서는, 상기 단계 (a)에서 얻은 패턴화된 배양 멤브레인을 친수화 시킨다. 보다 구체적으로, 상기 PDMS를 이용하여 단계 (a)에서 얻은 패턴화된 배양 멤브레인은 PDMS의 특성으로 인해 소수성을 나타내는바, 이를 친수화시키는 과정이 필요하며, 이를 위해 산소 플라즈마 처리를 하는 것이 바람직하다. 이 때, 산소 플라즈마 처리는 산소량 25 SCCM, 전력 40 W 및 40 SEC 조건하에서 수행되는 것이 바람직하다.In step (b), the patterned culture membrane obtained in step (a) is hydrophilized. More specifically, the patterned culture membrane obtained in step (a) using the PDMS shows a hydrophobicity due to the characteristics of the PDMS, it is necessary to hydrophilize it, it is preferable that the oxygen plasma treatment for this. At this time, the oxygen plasma treatment is preferably carried out under an oxygen amount of 25 SCCM, a power of 40 W, and 40 SEC.
단계 (c)에서는, 상기 단계 (b)에서 얻은 친수화된 배양 멤브레인을 피브로넥틴 (Fibronectin)으로 코팅하는 단계이다.In step (c), the hydrophilized culture membrane obtained in step (b) is coated with fibronectin.
이는, 상기 단계 (b) 진행 결과 충분한 친수화가 진행되지 않았는바, 상기 피브로넥틴 코팅을 통해서, 친수화를 증가시킬 수 있다. 이 때, 피브로넥틴은 상기 배양 멤브레인에 바로 코팅되지 않기 때문에 폴리-디-오르니틴 (Poly-DL-ornithine)을 이용하여 4시간 이상 전처리 한 후, 피브로넥틴으로 1시간 이상 코팅한다. 이로 인해 본 발명에 일실시예에 따른 유도만능줄기세포 배양 플레이트를 얻을 수 있다.This is because the sufficient hydrophilization did not proceed as a result of the step (b), through the fibronectin coating, it is possible to increase the hydrophilization. At this time, since the fibronectin is not directly coated on the culture membrane, it is pretreated with poly-DL-ornithine for 4 hours or more, and then coated with fibronectin for 1 hour or more. Thus, the induced pluripotent stem cell culture plate according to the embodiment of the present invention can be obtained.
단계 (d)에서는, 상기 단계 (c)에서 얻은 유도만능줄기세포 배양 플레이트에 유도만능줄기세포를 부착시키고, 계대 배양을 진행한다. In step (d), induced pluripotent stem cells are attached to the induced pluripotent stem cell culture plate obtained in step (c), and passaging is performed.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예]EXAMPLE
실시예 1. 본 발명에 따른 유도만능줄기세포 배양 플레이트 구현Example 1. Implementation of induced pluripotent stem cell culture plate according to the present invention
1-1. 편평한 형태의 패턴 1-1. Flat form pattern
멤브레인Membrane
(Flat pattern Membrane; FPM) 제작 및 표면 확인 (Flat pattern Membrane; FPM) fabrication and surface check
본 발명에 따른 유도만능줄기세포 배양 플레이트의 패턴에 따른 나노지형의 영향을 확인하기 위해서, 도 2a에 도시된 바와 같이, 패턴이 형성되지 않은 배양 멤브레인인 편평한 형태의 패턴 멤브레인을 비교 그룹으로 제작하였다.In order to confirm the effect of the nano-topography according to the pattern of the induced pluripotent stem cell culture plate according to the present invention, as shown in Figure 2a, a flat pattern pattern membrane, which is a culture membrane without a pattern is formed as a comparison group .
보다 구체적으로, 순수한 실리콘 웨이퍼상에 폴리디메틸실록산 (PDMS)를 붓고, 이 후, 스핀코터를 이용해서 스핀코팅을 진행한 후, 80℃ 온도로 12시간 가열하였다. 이 후, PDMS의 경화가 진행되어 완료되면, 편평한 형태의 배양 멤브레인 (FPM)을 얻었다.More specifically, polydimethylsiloxane (PDMS) was poured onto a pure silicon wafer, followed by spin coating using a spin coater, followed by heating to 80 ° C. for 12 hours. After that, when curing of PDMS proceeded and completed, a flat culture membrane (FPM) was obtained.
상기 FPM의 모양모사를 확인하기 위해서, 주사전자현미경 (Scanning electron microscopy; SEM) 및 원자간력현미경 (Atomic force micoscopy; AFM)을 이용하여 분석하였으며, 그 결과 도 2b에 도시된 바와 같이, SEM 및 AFM 이미지를 통해 매끄러운 표면을 가진 배양 멤브레인이 형성되는 것을 확인하였다.In order to confirm the shape simulation of the FPM, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were analyzed using SEM, and as a result, as shown in FIG. An AFM image confirmed that a culture membrane with a smooth surface was formed.
1-2. 불규칙한 패턴 1-2. Irregular pattern
멤브레인Membrane
(Irregular Pattern Membrane; Irregular Pattern Membrane;
IPMIPM
) 제작 및 표면 확인) Fabrication and surface check
실시예 1-1과 마찬가지로 본 발명에 따른 유도만능줄기세포 배양 플레이트의 패턴에 따른 나노지형의 영향을 확인하기 위해서, 도 3a에 도시된 바와 같이, 불규칙한 형태의 패턴 멤브레인 (IPM)을 제작하였다.As in Example 1-1, in order to confirm the effect of the nano-topography according to the pattern of the induced pluripotent stem cell culture plate according to the present invention, as shown in Figure 3a, an irregular pattern membrane (IPM) was prepared.
보다 구체적으로, 글라스 웨이퍼 (석영) 상에 플라이머를 뿌린 후, 2 시간 동안 건조시킨다. 이 후, 건조된 글라스 웨이퍼 위에 나노 프린트를 뿌리고, 상온에서 건조시켰으며, 건조시킨 글라스 웨이퍼 위에 PDMS를 도포한 후, 진공데시게이터에서 2시간 동안 넣어 놓았다. 이 후, 글라스 웨이퍼를 80℃ 온도로 12시간 가열하여 불규칙한 배양 멤브레인 (IPM)을 얻었으며, 이를 SEM 및 AFM의 이미지를 통해 확인한 결과, 도 3b에 도시된 바와 같이, 불규칙한 표면을 확인하였다. 또한, 도 3c에 도시된 바와 같이, 나노 표면의 높이가 평균 87.1 ㎚이고, 입자 패턴 사이의 거리는 226.2 내지 447.6 ㎚ 인 불규칙한 패턴 멤브레인이 형성되는 것을 확인하였다.More specifically, after spraying the primer on the glass wafer (quartz), it is dried for 2 hours. Thereafter, the nanoprint was sprayed onto the dried glass wafer, dried at room temperature, and then PDMS was applied to the dried glass wafer, and then placed in a vacuum desiccator for 2 hours. Thereafter, the glass wafer was heated to 80 ° C. for 12 hours to obtain an irregular culture membrane (IPM), which was confirmed through SEM and AFM images. As shown in FIG. 3B, the irregular surface was confirmed. In addition, as shown in FIG. 3C, it was confirmed that an irregular pattern membrane having an average surface height of 87.1 nm and a distance between particle patterns was 226.2 to 447.6 nm.
1-3. 반구형, 1-3. Hemisphere,
만곡진Curved line
호형, 파형 (Groove) 및 기둥형 (Post) 패턴 Arc, Groove, and Post Patterns
멤브레인Membrane
(Groove Pattern Membrane; GPM and Post Pattern Membrane: PPM) 제작 (Groove Pattern Membrane; GPM and Post Pattern Membrane: PPM)
본 발명에 따른 철부 및 요부가 복수개로 배열되어, Groove 및 Post 패턴을 형성하는 배양 멤브레인을 제작하기 위해서, E-beam 리소그래피 장비를 사용하여 포토 레지스트 패턴을 실리콘 웨이퍼에 형성 시킨 후, 패턴이 형성된 실리콘 웨이퍼를 Deep RIE (reactive ion etcher) 장비를 사용하여 실리콘 웨이퍼를 식각 (etching) 하였다. 다음으로 패턴화된 실리콘 웨이퍼를 실란 처리를 통하여, 표면을 소수성으로 변환시킨 후, 그 위에 PDMS를 부어 진공데시게이터에 4시간 동안 넣어 놓았다. 이 후, 실리콘 웨이퍼를 80℃ 온도로 12시간 가열한 결과, 도 4에 도시된 바와 같이, Groove 패턴 멤브레인 (GPM) 및 Post 패턴 멤브레인 (PPM)을 얻었다.In order to manufacture a culture membrane in which a plurality of convex portions and concave portions according to the present invention are formed to form a groove and a post pattern, a photoresist pattern is formed on a silicon wafer using an E-beam lithography apparatus, and then the patterned silicon is formed. The wafer was etched using a Deep RIE (reactive ion etcher) device. Next, the surface of the patterned silicon wafer was converted to hydrophobicity by silane treatment, and then PDMS was poured on it and placed in a vacuum desiccator for 4 hours. Thereafter, the silicon wafer was heated at a temperature of 80 ° C. for 12 hours, and as shown in FIG. 4, a Groove pattern membrane (GPM) and a Post pattern membrane (PPM) were obtained.
1-4. Groove 및 Post 패턴 1-4. Groove and Post Patterns
멤브레인Membrane
(Groove Pattern Membrane; Groove Pattern Membrane;
GPMGPM
and Post Pattern Membrane; PPM) 표면 확인 and Post Pattern Membrane; PPM) Surface Verification
GPM 및 PPM 패턴 멤브레인의 PDMS 개질 전에 master mold의 형태를 각각 SEM을 통하여 확인한 결과, 도 5a 및 도 5b에 도시된 바와 같이, GPM의 master mold의 경우, 폭이 239.7 ㎚, 높이가 98.5 ㎚, 사이 간격이 237.9 ㎚ 의 Groove 형태의 master mold를 확인하였으며, PPM의 경우, 폭이 237.2 ㎚, 높이 98.5 ㎚, 사이 간격이 237.2 ㎚ Post 형태의 master mold를 확인하였다.The shape of the master mold before the PDMS modification of the GPM and PPM pattern membranes was confirmed by SEM, respectively. As shown in FIGS. 5A and 5B, the GPM master mold had a width of 239.7 nm and a height of 98.5 nm. Groove-shaped master molds with a spacing of 237.9 nm were identified, and in the case of PPM, 237.2 nm in width, 98.5 nm in height, and 237.2 nm post-shaped master mold were found.
이 후, PDMS 개질을 진행한 결과, 도 5c 및 도 5d에 도시된 바와 같이, 각각 master mold의 음각과 양각의 방향이 반대로 되어진 GPM 및 PPM을 확인하였다. 보다 구체적으로, GPM의 경우 폭이 377.2 ㎚, 높이가 82.5 ㎚, 사이 간격이 161.1 ㎚ GPM을 확인하였으며, PPM의 경우 폭이 310 ㎚, 높이가 60.1 ㎚, 사이 간격이 100.7 ㎚ PPM을 확인하였다.Subsequently, as a result of the PDMS reforming, as shown in FIGS. 5C and 5D, the intaglio and embossed directions of the master mold were respectively reversed, and the GPM and the PPM were confirmed. More specifically, in the case of GPM, the width was 377.2 nm, the height was 82.5 nm, and the interval was 161.1 nm. The GPM was confirmed. In the case of PPM, the width was 310 nm, the height was 60.1 nm, and the interval was 100.7 nm.
1-5. 배양 멤브레인의 표면 거칠기 확인1-5. Check surface roughness of culture membrane
각 배양 멤브레인의 표면 거칠기를 확인하기 위하여, SEM을 통해 거칠기 값을 측정한 결과 하기 표 1과 같이 나타났다.In order to confirm the surface roughness of each culture membrane, the roughness value was measured as shown in Table 1 below.
[표 1]TABLE 1
그 결과, 상기 표 1에 도시된 바와 같이, IPM의 표면 거칠기 값이 가장 크게 측정된 것을 확인할 수 있었다.As a result, as shown in Table 1, it was confirmed that the surface roughness value of the IPM was measured the largest.
실시예 2. 배양 멤브레인의 표면 개질Example 2. Surface Modification of the Culture Membrane
2-1. 산소 플라즈마 처리를 이용한 친수화 처리2-1. Hydrophilization Treatment Using Oxygen Plasma Treatment
상기 실시예 1에서 제작된 배양 멤브레인은 모두 PDMS로 제작되어 소수성 (hydrophobic)을 띠고 있는바, 산소 플라즈마 처리를 통해서 친수성 표면으로 개질하는 친수화 처리를 진행하였다. 보다 구체적으로, 산소량 25 SCCM, 전력 40 W 및 시간 40 SEC의 조건하에서 산소 플라즈마 처리를 진행하였다.The culture membranes prepared in Example 1 were all made of PDMS to have hydrophobic, and were subjected to hydrophilization treatment to modify the hydrophilic surface through oxygen plasma treatment. More specifically, the oxygen plasma treatment was performed under conditions of an oxygen content of 25 SCCM, a power of 40 W, and a time of 40 SEC.
2-2. 개질제를 이용한 배양 멤브레인 표면 개질2-2. Surface Modification of Culture Membrane Using Modifiers
상기 실시예 2-1의 방법으로 친수화 처리를 진행한 결과 친수화가 충분히 이루어지지 않음을 확인하였으며, 이에, 본 발명의 일실시예에 따른 개질제를 이용하여 배양 멤브레인의 친수화를 증가시키고자 하였다. 보다 구체적으로, 산소 플라즈마 처리가 된 각 배양 멤브레인 표면을 개질제로 코팅을 진행하고, 접촉각을 측정하였다. 이 때, 개질제로는 피브로넥틴 (Fibronectin)을 사용하였다. As a result of the hydrophilization treatment according to the method of Example 2-1, it was confirmed that the hydrophilization was not sufficiently achieved. Accordingly, the modifier according to the embodiment of the present invention was intended to increase the hydrophilization of the culture membrane. . More specifically, the surface of each culture membrane subjected to oxygen plasma treatment was coated with a modifier and the contact angle was measured. At this time, fibronectin was used as a modifier.
그 결과, 도 6a에 도시된 바와 같이, 피브로넥틴 코팅전과 비교하여 PPM을 제외한 나머지 배양 멤브레인에서 접촉각이 감소하는 것을 확인하였다. 이로 인해, 피브로넥틴 코팅으로 인해 배양 멤브레인의 친수화를 증가시킬 수 있음을 확인하였다.As a result, as shown in Figure 6a, it was confirmed that the contact angle is reduced in the remaining culture membrane except for PPM compared to before fibronectin coating. This confirmed that the fibronectin coating can increase the hydrophilization of the culture membrane.
또한, 피브로넥틴 코팅이 패턴의 영향을 미치는지 확인하기 위해 코팅 후 배양 멤브레인을 SEM사진을 통하여 확인하였다. In addition, in order to confirm whether the fibronectin coating affects the pattern, the culture membrane after coating was confirmed through SEM photographs.
그 결과, 도 6b에 도시된 바와 같이, 배양 멤브레인의 패터닝에 크게 영향을 미치지 않았으나, FPM의 경우 코팅 전과 다르게 불규칙한 패턴이 형성되는 것을 확인하였다.As a result, as shown in Figure 6b, but did not significantly affect the patterning of the culture membrane, it was confirmed that the irregular pattern is formed in the case of FPM unlike before coating.
실시예Example
3. 본 발명에 따른 3. According to the present invention
유도만능줄기세포Induced pluripotent stem cells
배양 플레이트의 Of culture plate
유도만능줄기세포Induced pluripotent stem cells
배양능력 확인 Culture capacity check
3-1. 유도만능줄기세포 부착 및 증식 능력 확인3-1. Induced pluripotent stem cell adhesion and proliferation
각 피브로넥틴이 코팅된 패턴화된 배양 멤브레인이 유도만능줄기세포의 부착 및 증식능력에 미치는 영향을 확인하기 위해서, 상기 각 배양 멤브레인 상에서 유도만능줄기세포를 5일마다 계대배양을 유지하며 변화를 관찰하였으며, 계대 1 (passage 1; P 1), 계대 5 (P 5) 계대 10 (P 10)에서 유도만능줄기세포의 증식 모양을 확인하였다.In order to examine the effect of each fibronectin-coated patterned culture membrane on the adhesion and proliferation ability of induced pluripotent stem cells, the induced pluripotent stem cells were passaged every 5 days and observed changes. Proliferation patterns of induced pluripotent stem cells were observed at passage 1 (passage 1; P 1) and passage 5 (P 5).
그 결과 도 7의 A에 도시된 바와 같이, GPM 및 PPM 상에서 유도만능줄기세포는, P 1에서 콜로니의 형태로 뭉쳐져서 자라며 제대로 증식이 되지 않는 모습을 보였으나, 계대가 진행될수록 콜로니를 이루지 않고, 정상적으로 증식되어 배양 멤브레인을 꽉 채우는 모습을 확인하였다. As a result, as shown in FIG. 7A, induced pluripotent stem cells on GPM and PPM were aggregated in the form of colonies in P 1 and grew and did not proliferate properly, but did not form colonies as passage was progressed. As a result, the cells were grown normally and filled with culture membranes.
또한, 각 배양 멤브레인이 유도만능줄기세포의 증식에 미치는 영향을 확인하기 위해서, Population doubling level (PDL) 및 total PDL을 통하여 확인하였다.In addition, in order to confirm the effect of each culture membrane on the proliferation of induced pluripotent stem cells, it was confirmed through the Population doubling level (PDL) and total PDL.
그 결과 도 7의 B에 도시된 바와 같이, total PDL은 각 배양 멤브레인에서 큰 변화가 없었지만, P 1에서의 PDL은 FPM과 비교할 때, GPM 및 PPM에서 다소 낮은 것을 확인하였다.As a result, as shown in FIG. 7B, the total PDL did not change significantly in each culture membrane, but the PDL at P 1 was found to be somewhat lower in GPM and PPM compared to FPM.
더욱이, 각 배양 멤브레인이 유도만능줄기세포의 증식에 미치는 영향을 확인하기 위해서, 세포 증식의 마커인 Ki67의 mRNA 발현량과 면역형광염색을 통하여 확인하였다. Furthermore, in order to confirm the effect of each culture membrane on the proliferation of induced pluripotent stem cells, it was confirmed through mRNA expression and immunofluorescence staining of Ki67, a marker of cell proliferation.
그 결과 도 7의 C 및 D에 도시된 바와 같이, 계대가 계속 진행됨에 따라서, FPM 및 IPM에서 Ki67의 발현량은 점차 감소하고 있으나, GPM 및 PPM에서는 유도만능줄기세포의 Ki67의 발현량이 떨어지지 않았으며, GPM의 경우 오히려 계대가 진행될수록 발현량이 증가되는 것을 확인하였다.As a result, as shown in C and D of FIG. 7, as the passage continues, the expression level of Ki67 in FPM and IPM is gradually decreased, but the expression level of Ki67 in induced pluripotent stem cells did not drop in GPM and PPM. And, in the case of GPM, as the passage progressed, it was confirmed that the expression level increased.
3-2. 줄기세포성 유지 능력 확인3-2. Stem cell maintenance ability
각 배양 멤브레인의 유도만능줄기세포의 계속된 계대배양시 미분화상태를 유지하는 능력을 확인하기 위해서, 미분화 배아줄기세포 마커인 Oct3/4 및 Nanog의 mRNA 발현량 및 면역화학염색을 진행하였다.In order to confirm the ability of each culture membrane to maintain the undifferentiated state during continuous passage of induced pluripotent stem cells, mRNA expression and immunochemical staining of the undifferentiated embryonic stem cell markers Oct3 / 4 and Nanog were performed.
그 결과 도 8의 A, B 및 C에 도시된 바와 같이, P 1에서는 큰 차이가 없었으나, 계대가 진행되면서 GPM 및 PPM에서 mRNA 발현량 및 면역화학염색을 통한 발현세포가 크게 증가하는 것을 확인하였다.As a result, as shown in A, B and C of FIG. 8, there was no significant difference in P 1, but as passage progressed, it was confirmed that the mRNA expression amount and the cells expressed through the immunochemical staining were significantly increased in GPM and PPM. It was.
이를 통해, 본 발명에 따른 유도만능줄기세포 배양 플레이트를 이용한 계대배양은 유도만능줄기세포 줄기세포성 유지에 효과적이며, 특히, 규칙적인 패턴, 보다 구체적으로 GPM 및 PPM에서 그 효과가 크게 나타나는 것을 확인하였다.Through this, subcultures using the induced pluripotent stem cell culture plate according to the present invention is effective in maintaining induced pluripotent stem cell stem cell activity, in particular, the regular pattern, more specifically confirmed that the effect is large in GPM and PPM It was.
3-3. 유도만능줄기세포의 형태학적 모양 확인3-3. Morphological Appearance of Induced Pluripotent Stem Cells
각 배양 멤브레인의 유도만능줄기세포의 세포 부착시 세포의 형태학적 모양에 미치는 영향을 확인하기 위해서, P 5에서 SEM 및 AFM을 이용하여 확인하였다.In order to determine the effect of the induced pluripotent stem cells on the morphological shape of the cells in the cell adhesion of each culture membrane, it was confirmed using SEM and AFM at P 5.
그 결과 도 9의 A 및 B에 도시된 바와 같이, FPM 및 IPM에서는 많은 cell fiber들이 뻗어나가면서 부착되어 있는 모습을 확인하였으며, GPM 및 PPM에서는 FPM과 비교하여 상대적으로 cell fiber들이 뻗어나가지 못한 뭉툭한 모양으로 부착되어 있음을 확인하였다.As a result, as shown in Figures A and B of Figure 9, in the FPM and IPM it was confirmed that a lot of cell fibers are attached and stretched, and in the GPM and PPM compared to the FPM relatively blunt shape that the cell fibers did not extend It was confirmed that it is attached.
3-4. 유도만능줄기세포의 부착 관계 신호전달 인자 확인3-4. Identification of Adhesion-Related Signaling Factors in Induced Pluripotent Stem Cells
본 발명에 따른 유도만능줄기세포 배양 플레이트 상에서 배양된 유도만능줄기세포를 이용하여 세포 부착과 관련된 인자에 있어서, 인테그린 수용체를 통해서 시작된 외부 신호를 전달하고 증폭시켜 내부로 전달해 주는 인자, 예컨대 Focal adhesion kinase (FAK), a-actinin, Paxillin, Vinculin, Talin 및 Zyxin 등의 단백질의 발현량을 real-time PCR 및 western blot을 통해 확인하였다.Factors related to cell adhesion using induced pluripotent stem cells cultured on induced pluripotent stem cell culture plates according to the present invention, such as Focal adhesion kinase, which transmits and amplifies external signals initiated through integrin receptors (FAK), a-actinin, Paxillin, Vinculin, Talin and Zyxin expression levels of proteins were confirmed by real-time PCR and western blot.
그 결과 도 10의 A 및 B에 도시된 바와 같이, a-actinin, Vinculin 및 Paxillin은 배양 멤브레인에서의 계대가 진행될수록 전체적인 발현량이 증가하였으며, FAK는 GPM 및 PPM에서만 다른 인자들에 비해 빠르게 P 6에서 증가하는 것을 확인하였으며, Talin 및 Zyxin은 다른 부착 인자들과는 다른 발현 양상을 보였다.As a result, as shown in A and B of Figure 10, a-actinin, Vinculin and Paxillin increased the overall expression as passage in the culture membrane, FAK was faster than other factors only in GPM and PPM P 6 In addition, Talin and Zyxin showed different expressions from other adhesion factors.
또한, p-FAK의 protein 발현량이 P 6와 P 10의 경우에는 다소 높게 나타나는 것으로 확인되었으며, FAK의 인산화 증가와 더불어 FAK와 결합하여 세포의 주요 생리현상을 조절하는 것으로 알려진 Paxillin의 인산화도 배양 멤브레인에서의 초기배양시에는 FPM에서 높았으나, 계대가 진행됨에 따라 GPM 및 PPM에서 높게 나타나는 것을 확인하였다.In addition, the protein expression level of p-FAK was found to be slightly higher in P 6 and P 10. In addition to the increased phosphorylation of FAK, the phosphorylation of Paxillin, which is known to regulate the major physiological phenomena of cells by binding to FAK, was also cultured. The initial culture at was high in the FPM, but as the passage progressed it was confirmed that the higher in the GPM and PPM.
3-5. 유도만능줄기세포의 세포골격 변화 확인3-5. Confirmation of cytoskeletal changes of induced pluripotent stem cells
세포외기질을 통한 물리적 자극에 대한 힘의 차이를 인식하고 세포의 행동 방향을 결정짓는 최종 단계에서 세포골격이 매우 중요한 역할을 하는바, 세포골격의 변화를 Phalloidin 염색을 통해 확인하였다.The cytoskeleton plays a very important role in the final step of recognizing the difference in force on the physical stimulation through extracellular matrix and determining the behavior of the cell. The change in the cytoskeleton was confirmed by Phalloidin staining.
그 결과 도 11에 도시된 바와 같이, Phalloidin 및 Vinculin의 면역화학염색 결과, 전체적인 cytoskeleton의 변화는 보이지 않았으나, Vinculin은 GPM 및 PPM에서 더 많이 발현되며, P 1에 비해 P 10의 유도만능줄기세포에서 많이 발현되는 것을 확인하였다.As a result, as shown in Figure 11, immunohistochemical staining of Phalloidin and Vinculin did not show a change in the overall cytoskeleton, Vinculin is more expressed in GPM and PPM, compared to P 1 in induced pluripotent stem cells It was confirmed that a lot of expression.
이를 통해, 규칙적인 패턴의 배양 멤브레인, 보다 구체적으로 GPM 및 PPM에서의 유도만능줄기세포의 계대 배양의 계속된 진행은 세포 부착의 모양에 영향을 미치지 않으면서, Focal adhesion molecules의 발현도 증가시키는 것을 확인하였다.This suggests that continued progression of subcultures of induced pluripotent stem cells in a regular pattern of culture membranes, more specifically in GPM and PPM, also increases the expression of Focal adhesion molecules without affecting the appearance of cell adhesion. Confirmed.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
본 발명에 따른 유도만능줄기세포 배양 플레이트는, 생체내에서의 물리적 미세환경 즉, 세포외기질의 영향을 나노지형 (nanotopography)을 이용하여 재현하는 패턴화된 배양 멤브레인 및 상기 배양 멤브레인 표면에 코팅된 개질제를 포함함으로서, 상기 배양 플레이트에 사람유도만능줄기세포를 부착하여 배양하였을 때, 종래의 줄기세포의 계대배양 과정 중에서 손실되는 줄기세포성 유지의 문제를 해결하였는바, 증식 능력 및 부착 능력이 보다 우수하고, 미분화 유지능이 향상된 사람유도만능줄기세포를 다량 획득할 것으로 기대된다.Induced pluripotent stem cell culture plate according to the present invention is a patterned culture membrane and a modifier coated on the surface of the culture membrane to reproduce the effects of physical microenvironment, that is, extracellular matrix in vivo using nanotopography By including, when the human induced pluripotent stem cells attached to the culture plate and cultured, the problem of stem cell maintenance that is lost during the passage of the conventional stem cells, bar proliferation ability and adhesion ability is more excellent In addition, it is expected to obtain a large amount of human induced pluripotent stem cells with improved undifferentiated maintenance ability.
Claims (7)
- 유도만능줄기세포 배양 플레이트에 있어서,In induced pluripotent stem cell culture plate,300-400㎚의 직경을 갖는 철부 및 100-300㎚의 직경을 갖는 요부가 복수개로 배열되어 패턴을 형성하는 배양 멤브레인; 및A culture membrane in which a convex portion having a diameter of 300-400 nm and a concave portion having a diameter of 100-300 nm are arranged in a plurality to form a pattern; And상기 배양 멤브레인 표면에 코팅된 개질제를 포함하는, 유도만능줄기세포 배양 플레이트.Induced pluripotent stem cell culture plate comprising a modifier coated on the culture membrane surface.
- 제1항에 있어서, 상기 배양 멤브레인은, 폴리디메틸실록산(PDMS)으로 이루어진 것을 특징으로 하는, 유도만능줄기세포 배양 플레이트.The induced pluripotent stem cell culture plate according to claim 1, wherein the culture membrane is made of polydimethylsiloxane (PDMS).
- 제1항에 있어서, 상기 개질제는 피브로넥틴 (Fibronectin)인 것을 특징으로 하는, 유도만능줄기세포 배양 플레이트.According to claim 1, wherein the modifier is fibronectin (Fibronectin), characterized in that, induced pluripotent stem cell culture plate.
- 제1항에 있어서, 상기 철부 및 요부는, 반구형, 만곡진 호형, 파형 또는 기둥형으로 이루어지는 것을 특징으로 하는, 유도만능줄기세포 배양 플레이트.The induced pluripotent stem cell culture plate according to claim 1, wherein the convex portion and the concave portion are formed in a hemispherical shape, a curved arc shape, a wave shape, or a pillar shape.
- 제1항에 있어서, 상기 배양 플레이트는, 유도만능줄기세포 (Induced pluripotent stem cells)의 부착 및 증식능을 향상시키는 것을 특징으로 하는, 유도만능줄기세포 배양 플레이트.According to claim 1, The culture plate, induced pluripotent stem cells (Induced pluripotent stem cells), characterized in that to improve the adhesion and proliferation, induced pluripotent stem cell culture plate.
- 제1항에 있어서, 상기 배양 플레이트는, 유도만능줄기세포 (Induced pluripotent stem cells)의 미분화 유지능을 향상시키는 것을 특징으로 하는, 유도만능줄기세포 배양 플레이트.The induced pluripotent stem cell culture plate according to claim 1, wherein the culture plate improves undifferentiated maintenance ability of induced pluripotent stem cells.
- 하기의 단계를 포함하는 유도만능줄기세포 배양 플레이트를 이용한 유도만능줄기세포 배양 방법:Induced pluripotent stem cell culture method using induced pluripotent stem cell culture plate comprising the following steps:(a) 300-400㎚의 직경을 갖는 복수개의 철부 및 100-300㎚의 직경을 갖는 요부가 복수개로 배열되어 패턴을 형성하는 배양 멤브레인을 기판 상면에 배치시키는 단계;(a) disposing a plurality of convex portions having a diameter of 300-400 nm and a plurality of concave portions having a diameter of 100-300 nm arranged on a top surface of the substrate to form a pattern;(b) 상기 배양 멤브레인 표면을 친수성으로 개질시키는 단계;(b) modifying the culture membrane surface to be hydrophilic;(c) 개질된 상기 배양 멤브레인 표면을 개질제로 코팅시키는 단계; 및(c) coating the modified culture membrane surface with a modifier; And(d) 상기 (c) 단계에서 제조된 배양 테이블에 유도만능줄기세포를 부착하여 배양하는 단계.(d) culturing by attaching induced pluripotent stem cells to the culture table prepared in step (c).
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