KR20140129841A - A screening method for the optimal surface structure of embryonic stem cell culture using a cell culture plate comprising gradient nanopattern - Google Patents

Abstract

The present invention relates to a method for screening surface structures suitable to culture embryonic stem cells which comprises a step of culturing cells in a cell culture container containing a gradient nanocolumn array of which the diameter or spacing uniformly changes; a cell culture container used in the screening method and having nanocolumns with regularly increasing diameters uniformly arranged at regularly decreasing intervals; and a method for culturing embryonic stem cells while maintaining stemness without feeder cells which comprises a step of culturing cells in a cell culture container containing the nanocolumns selected by the screening method. The cell culture container comprising nanocolumns with regularly increasing diameters uniformly arranged at regularly decreasing intervals of the present invention can provide various surface morphologies in a single culture container, and thus can simultaneously monitor effects of the surface morphology change on cellular characteristics in a single culture conducted under the same conditions, thereby selecting optimum surface morphologies suitable for cell culture. The method for culturing human embryonic stem cells selected through the screening method can achieve the proliferation of the embryonic stem cells while maintaining stemness without needing to coat expensive extracellular substrates or use feeder cells.

Description

[0001] The present invention relates to a method for screening a surface structure suitable for culturing embryonic stem cells using a culture vessel containing a nano-gradient pattern,

The present invention provides a method for screening a surface structure suitable for embryonic stem cell culture comprising culturing a cell in a cell culture container containing a gradient nano pillar array having a constant diameter or diameter, A cell culture container containing nano pillars uniformly arranged at intervals such that the nano pillars having a constantly increasing diameter used in the screening method are uniformly arranged at an interval decreasing uniformly, and a cell culture comprising nanoparticles of the conditions selected by the screening method And culturing the embryonic stem cells while maintaining stem cell morphology without supporting cells comprising culturing the cells in a container.

In the 21st century, biotechnology is aiming at human welfare, suggesting the possibility of new solutions to food, environment and health problems. Recently, stem cell utilization technology is emerging as a new field of intractable disease treatment. Until now, organ transplantation and gene therapy have been proposed for the treatment of incurable diseases, but practical use has been limited due to lack of immunity, lack of supply or lack of knowledge about genes.

Thus, interest in stem cell research has been heightened, and it has been recognized that all stem cells capable of forming all organs through proliferation and differentiation can solve root diseases as well as treatment of most diseases. In addition, many scientists have proposed a variety of stem cell applications, ranging from treatment of intractable Parkinson's disease, various cancers, diabetes and spinal cord injury to almost all organ regeneration.

Stem cells are cells capable of self-replication and capable of differentiating into two or more cells. They are totipotent stem cells, pluripotent stem cells, multipotential stem cells multipotent stem cells (MSCs).

Allogeneic stem cells are pluripotent cells that can develop into one complete organism. After the fertilization of the oocyte and spermatozoa, cells up to 8 years of age have this property. When these cells are isolated and transplanted into the uterus, one complete It can occur as an entity. Pre-differentiation stem cells originate from the inner cell mass located inside the blastocyst, which appears 4 to 5 days after fertilization. The stem cells are derived from various cells and tissues derived from ectoderm, mesoderm, and endodermal layer. It is called embryonic stem cells and differentiates into a variety of different tissue cells but does not form new life forms.

The pluripotent stem cells were first isolated from adult bone marrow (Y. Jiang et al., Nature, 418: 41, 2002) and subsequently identified in many other adult tissues (CM Verfaillie, Trends Cell Biol., 12: 502, 2002). In other words, bone marrow is the most widely known source of stem cells, but multipotential stem cells have also been identified from skin, blood vessels, muscle and brain (JG Toma et al., Nat. Cell Biol., 3: 778, 2001; Sampaolesi et al., Science, 301: 487, 2003; Y. Jiang et al., Exp. Hematol., 30: 896,2002). However, stem cells in adult tissues such as bone marrow are very rarely present, and these cells are difficult to culture without inducing differentiation, and it is difficult to cultivate them without specific screened media.

The fate of the stem cells is determined by a number of factors that constitute complexity and kinetics in vivo niche. Attempts to simulate the microenvironment of a cell during in vitro culture depend on the use of soluble factors (e.g., growth factors, cytokines, small molecules, nutrients, etc.). However, accumulated evidence indicates that, in addition to the biochemical environment, the physical interactions between stem cells and underlying basement membrane serve as important mediators for a variety of cellular behaviors.

The basement membrane is extracellular matrices (ECMs) that provide a substrate for the survival, proliferation and differentiation of adhesion-dependent stem cells. Topological studies have shown that the basement membrane is porous at the nano-level and is formed by interwinding fibrous proteins such as laminin and collagen. Of pores of various sizes. Recent advances in nanotechnology, such as self-assembly and lithography, have produced various types of nanoscale surface features to study cell-nanotopography interactions for stem cell behavior It was possible. For example, the spatial arrangement of the adhesive dots can modulate cell diffusion by altering the assembly of focal adhesions and cytoskeletal organization, and can be used for various types of nanostructures (e.g., line gratings ), Columns, tubes and porosity) can affect the maintenance, differentiation and proliferation of various types of stem cells.

In general, embryonic stem cells are co-cultured with mouse or human-derived supporting cells (usually fibroblasts). The supporting cells provide secretory factors, extracellular matrix (ECM), and cell contact so that the embryonic stem cells do not lose stem cell function but remain undifferentiated. However, the use of the supporting cells has been a factor of obstructing the clinical application of human embryonic stem cells because of the risk of cross contamination such as pathogens of animal are transferred to human embryonic stem cells. Therefore, research is underway to find out an embryonic stem cell culture system that does not use such supporting cells. As part of this effort, cultured embryonic stem cells can be cultured in a conditioned medium (CM) or other growth factors such as medium cultured with mouse or human supporting cells, and other signaling Lt; RTI ID = 0.0 > factor. ≪ / RTI >

Conventionally, as another method of culturing embryonic stem cells while maintaining stem cell function without supporting cells, mTeSR medium or the like was used to coat the surface of the plate with matrigel at a low temperature. However, in the case of using Matrigel, there is a complicated problem that a reagent treatment process for maintaining the surface of the plate at a low temperature and removing matrigel is added during the subculturing of embryonic stem cells. In addition, mTeSR medium (feeder-free media, Stem Cell Technologie) requires an expensive protein coating (matrigel, BD Science). In addition, cost of the culture method is also problematic since the mTeSR medium having a high unit price needs to be exchanged every day.

Therefore, the inventors of the present invention have sought to find an optimal condition for culturing embryonic stem cells while maintaining stem cell function without supporting cells. As a result, it has been found that a constantly increasing diameter of the nanoparticles When the cells were cultured using a cell culture container containing nanoparticles uniformly arranged in the same manner as that of Example 1, it was confirmed that the expression of the differentiation-ability marker was maintained at a higher level in a specific diameter range, The present invention has been accomplished by confirming that the container can be used for searching for optimal surface structure for differentiation, proliferation and maintenance of stem cells.

One object of the present invention is to provide a method for screening a surface structure suitable for cell culture comprising culturing adherent cells in a cell culture container containing a gradient nano pillar array in which diameter or spacing is constantly changed To provide a method to do so.

It is another object of the present invention to provide a cell culture container comprising nano pillars uniformly arranged at intervals such that the nano pillars having a constantly increasing diameter used in the screening method are uniformly reduced.

It is still another object of the present invention to provide a cell culture for stem cell function maintenance comprising nano pillars uniformly arranged at intervals of 320 nm to 250 nm with nano pillars having diameters of 120 nm to 170 nm selected by the screening method To provide a container.

It is still another object of the present invention to provide a method for culturing embryonic stem cells while maintaining stem cell function without supporting cells comprising culturing the cells in the cell culture container.

According to one aspect of the present invention, there is provided an embryonic stem cell (embryonic stem cell) comprising a cell culture container comprising a cell culture container containing a gradient nano pillar array having a constant diameter or diameter A method for screening a surface structure suitable for culturing is provided.

In another aspect, the present invention provides a cell culture container comprising nano pillars uniformly arranged at intervals such that nano pillars having a constantly increasing diameter used in the screening method are uniformly reduced.

The method for screening a surface structure suitable for embryonic stem cell culture according to the present invention may be carried out using a cell culture container prepared to include nanoparticles having a diameter and an interval that change at the same time. The homogeneous arrangement of the nanopillar poles having the same diameter and spacing at the same time was defined as a " dual gradient nanopattern "or abbreviated as a " gradient nanopattern (G-Np) ".

The term "gradient " of the present invention means a constant and regular change. The double nano-gradient pattern introduced into the cell culture container according to the present invention may be designed so that the diameter and the spacing increase at the same time, the diameter and the spacing decrease at the same time, or the diameter increases but the spacing decreases. Preferably, the present invention is used to describe the diameter of a nanopillar increasing at a constant rate and the distance between the nanopillars decreasing constantly.

The diameter of the nano-pillars means the diameter measured assuming that the upper part of the nano-pillars is a circle of similar size. The distance from the center of one nano-pillar to the center of the nearest neighboring nano-pillar is measured, Can be calculated as the difference value minus the radius.

Preferably, the nano-gradient pattern comprising nanocrystals that increase in diameter and decrease in spacing is controlled by controlling the exposure time to the acid solution so that anionic oxidation, including nanoporous pores with diameters increasing from one end to another, And can be produced using aluminum (anodic aluminum oxide; AAO). For example, the anionic aluminum oxide may be prepared by anodizing an aluminum plate in a phosphoric acid solution and etching it with a chromic acid solution. The resulting AAO with aligned pores can be anodized again and processed in a phosphoric acid solution with a gradual increase in immersion time to produce AAO molds with constant pore diameters. Finally, the prepared AAO template can be manufactured by thermal imprinting on a substrate of a material which can be used as a cell culture container without cytotoxicity such as polystyrene. The manufacturing process may further include coating a single layer of heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane (HDFS) on the AAO template by self-assembly prior to thermal imprinting.

The immersing time in the phosphoric acid solution for preparing the AAO template in which the pore size is constantly increased is preferably from 0 minutes to 500 minutes, but is not limited thereto.

Since the substrate for producing the nano-gradient pattern with the heat angle of the AAO including the pores increasing in the diameter of the constant diameter is intended for cell culture for the purpose of the present invention, a biocompatible polymer material . ≪ / RTI > Preferably, it may be a polystyrene material which is generally used as a material of a cell culture container used for cell culture, and a commercially available polystyrene cell culture container may be directly used, but is not limited thereto. In the case of using a polystyrene substrate, the thermal imprint can be performed by heating the substrate at 100 to 150 DEG C for 7 to 13 minutes and compressing the substrate at 2.5 to 3.5 bar for 80 to 120 seconds.

Preferably, the cell culture vessel according to the present invention may be prepared so that the nanopillars having an increasing diameter gradient of 100 nm to 400 nm comprise a double nano-gradient pattern arranged at decreasing intervals of 350 nm to 50 nm. For example, at one end of the nano-gradient pattern, nanoparticles having an average diameter of ~ 120 nm are arranged at intervals of ~ 320 nm, and nanopillars having an average diameter of ~ 230 nm are arranged at intervals of ~220 nm near the center of the pattern. At the other end of the pattern, the diameters of the nanoparticles linearly increase as the nanoparticles of ~ 350 nm in average diameter are arranged at ~ 80 nm intervals from one end of the pattern to the other end, and the spacing decreases linearly Lt; RTI ID = 0.0 > diameter < / RTI > At this time, the height of the nanopillar may be 100 nm to 1 mm, but is not limited thereto. Preferably from 100 nm to 1000 nm.

The term "adhesive " of the present invention refers to the property that a cell binds to a surface, an extracellular matrix or other cells through a cell adhesion molecule. Such cell attachment molecules include selectins, integrins, and cadherins. Appropriate cell adhesion is an essential characteristic for the maintenance of the multicellular structure. The cell adhesion may connect the cytoplasm of the cell and may be involved in signal transduction. Due to the adherence of these cells to the surface, they can be affected by the morphology of the junction surface.

The cell culture container containing the nanopillar array of the present invention can be coated with a coating material known in the art in order to facilitate cell adhesion. Preferably, the coating material may be collagen, alginate, poly-D-lysine or agar, but may be a material capable of enhancing cell adhesion to the culture dish without exhibiting cytotoxicity You can use it without limitation.

The method of the present invention may further include the step of analyzing the characteristics of the embryonic stem cells collected in the region including the nanoparticles of different diameters and intervals of the diameter gradient nanopillar array. And the regions showing the most preferable cell characteristics can be selected by comparing and analyzing the results collected through the above characteristics analysis.

The term "embryonic stem cells " of the present invention is pluripotent stem cells derived from inner cell mass (ICM) of blastocyst in early embryonic development. Human embryos reach blastocyst stage consisting of 50 to 150 cells 4 to 5 days after fertilization. An embryoblast or an inner cell mass can be obtained by destroying a modified human embryo. The obtained embryonic stem cells have a size of about 14 μm for human and about 8 μm for mouse. Two distinctive features of the embryonic stem cells are 1) the ability to differentiate and 2) the ability to replicate infinitely. Embryonic stem cells have differentiation potential and can be differentiated into all three primary germ layers of ectoderm, endoderm and mesoderm. This includes more than 220 cell types in the adult. The ability to differentiate is a characteristic of embryonic stem cells distinguished from adult stem cells found in adults. For example, embryonic stem cells can be differentiated into all cell types in the body, but adult stem cells are multipotent and can only be differentiated into a limited number of cell types. The embryonic stem cells can also self-proliferate indefinitely under limited conditions. Therefore, the embryonic stem cells can be usefully used in research and reproduction medicine.

Therefore, the step of analyzing the characteristics of the collected embryonic stem cells can be performed by measuring the expression of the pre-differentiability marker. At this time, it is preferable that the pre-multipoint marker is Oct4, but it is not limited thereto.

Octan-binding transcription factor 4 (Oct4) is a protein, also known as POU5F1 (POU domain, class 5, transcription factor 1), which is the homeodomain transcription factor of the POU family. The protein is critically involved in the self-renewal of undifferentiated embryonic stem cells. For the above-mentioned reason, the protein is frequently used as a marker for undifferentiated cells, and monitoring of the marker is very important for understanding the differentiation state of the cells.

In addition to the cell characteristics of the stem cells described above, the undifferentiated embryonic stem cells form a dense colony and have morphological characteristics of being proliferated and maintained. Each of the embryonic stem cells forming the dense colonies is characterized by having a high nuclear / cytoplasmic ratio. Therefore, the step of analyzing the characteristics of the cell may be performed by further measuring the expression of a cell adhesion marker, a cytoskeletal marker or a tumor marker, and the cell adhesion marker may be E-cadherin, , The cytoskeletal marker may be phalloidin, and the lesion attachment marker may be vinculin.

The step of analyzing the cellular and / or morphological characteristics of the collected embryonic stem cells can be carried out through immunoassay using an antibody specifically binding to the markers. The antibodies may be fluorescence-labeled to facilitate detection, and two or more markers may be detected at the same time when fluorescent materials emitting light at different wavelengths are labeled. Further, in performing the immunoassay, the position and / or distribution of the cells can be confirmed by staining the DNA nuclei using DAPI (4 ', 6-diamidino-2-phenylindole). For the purpose of the present invention, the DAPI is used to measure the position and / or shape of a cell. Therefore, the DAPI can be used in place of a substance used for staining nuclei, cytoplasm or mitochondria well known in the art. Non-limiting examples of these include methylene blue, acetocarmine, toluidine blue, hematoxylin or Hoechst, which stain nuclei, eosin, which stains cytosol, Such as rhodamine 123, MitoTracker, janus green B, tetrazolium salt, which stains mitochondria, such as crystal violet or orange G, , DASPMI (Dimethylaminostyrylmethylpyridiniumiodine), DASPEI (2- (4- (dimethylamino) styryl) -N-Ethylpyridinium Iodide), DiOC6 (3,3'-dihexyloxacarbocyanine iodide), DiOC7 (3,3'- Diheptyloxacarbocyanine Iodide) (5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-benzimidazolylcarbocyanine chloride).

The term " epithelial cadherin (E-cadherin) "is a protein also known as cadherin-1, CAM 120/80 or uvomorulin and is a tumor suppressor gene It is encoded by the CDH1 gene. The E-cadherin is a representative member of the cadherin superfamily. The encoded protein is composed of five extracellular cadherin repeats EC1 to EC5 present in the extracellular domain, a transmembrane region ) And an intracellular domain that binds to p120-catenin and beta-catenin. The intracellular domain contains a highly phosphorylated region essential for beta-catenin binding. The beta -catenin also binds alpha-catenin, which is involved in the regulation of cytoskeletal filaments, including actin. Thus, the E-cadherin contains cell-cell junctions and is present adjacent to cytoskeletal filaments containing actin. The E-cadherin is first expressed in the second stage of mammalian development and is phosphorylated at the 8th cell stage to induce a dense structure.

The above-mentioned "Paloidine" is a kind of toxin derived from a highly toxic white mushroom known as phallotoxin and specifically binds to F-actin. In particular, Paloinden binds to the boundaries between F-actin subunits where adjacent subunits engage with each other. Therefore, phalloidin labeled with the phosphor can be used as a useful tool for confirming the distribution of F-Actin in cells. On the other hand, the above-mentioned "F-actin" is a linear polymer microfilament essential for cell functions such as cell shrinkage and cell mobility during cell division, and is a main component constituting the cytoskeleton. Therefore, the F-actin is mainly distributed around the cell and / or at the boundary between cells and cells, and is involved in establishment and maintenance of cell junction and cell morphology, cell signaling, and the like. Thus, the paloidin staining may provide information on cell-cell boundaries and cell morphology.

The above-mentioned "vinculin" is a membrane-cytoskeletal protein in a focal adhesion plaque that participates in the binding of an integrin adhesion molecule to the actin cytoskeleton. To 30% sequence similarity. Talline or alpha-actinin, thereby changing the form and binding capacity of the vaculin. The vinculin is a cytoskeletal protein that is involved in cell-cell and cell-matrix junctions and is thought to act as one of the proteins involved in fixing F-actin to the membrane.

In a specific example of the present invention, cell nucleus staining by DAPI and Paloidin, which is a cytoskeletal forming marker, were simultaneously observed. On the control panel, it was confirmed that F-actin, which is a cytoskeletal protein, was diffused far from the nucleus, and the nuclear / cytoplasmic ratio was greatly decreased (FIGS. 6A to 6C). On the other hand, the cells cultured in the culture vessel having the nano-gradient pattern according to the present invention are located close to the nucleus and cytoskeletal protein F-actin, although there is a slight difference depending on the sampling region, And the cytoplasmic ratio was maintained at a very high level (Figs. 6A, 6B and 6D to 6F). In the case of E-cadherin involved in the formation of cytoskeletal filaments, in the case of cells cultured in a culture vessel containing the nano-gradient pattern according to the present invention, the cell-skeletal protein, F-actin, , Whereas the cells cultured on the face of the control panel showed not only staining at the cell boundary but also significantly decreased expression (FIGS. 6C to 6F).

Preferably, the method for screening a surface structure suitable for cell culture according to the present invention is useful for searching for a condition for culturing embryonic stem cells while maintaining stem cell function without supporting cells.

The term "stemness" of the present invention is a term used to describe the characteristics of stem cells distinguished from somatic cells capable of differentiating into two or more cells while having self-replicating ability. That is, the stem cell function may be a term used to express the characteristics of a stem cell, which is a cell having both excellent self-replicating ability through cell division and differentiating ability to differentiate into various cells.

In a specific embodiment of the present invention, the nano-gradient pattern is divided into three different regions according to the diameter of the nanoparticles, and the morphology of human embryonic stem cells cultured without supporting cells on each region and the expression of Oct4, 4C). It was confirmed that the cells cultured in the region containing the nanoparticles in the diameter range can form colonies denser than those cultured in the other regions and maintain the expression rate of Oct at a higher level (Fig. 4C). For example, when human embryonic stem cells were cultured without supporting cells in a region containing nanoparticles having a diameter ranging from 120 nm to 170 nm (low region: Low), it was confirmed that the expression rate of Oct4 was maintained at a level exceeding 90% Respectively.

In another aspect, the present invention provides a cell culture container for maintaining stem cell performance comprising nano pillars having nano pillars having diameters of 120 nm to 170 nm uniformly arranged at intervals of 320 nm to 250 nm. The nanopattern of the cell culture container was screened by the screening method according to the present invention. The nanopattern corresponds to an optimal condition for maintaining stem cell function when embryonic stem cells are cultured without supporting cells.

Accordingly, the present invention provides a method for culturing embryonic stem cells while maintaining stem cell function without supporting cells comprising culturing the cells in the cell culture container.

As described in the above screening method, when embryonic stem cells are cultured without supporting cells, the cells cultured in a specific region of the nano-gradient pattern of the present invention exhibit significantly higher levels of Oct4, Respectively. Therefore, by selecting the portion having the diameter and the interval of the nanoparticles in the range where the expression of Oct4 selected by the screening method is maintained at the highest level, a cell culture container containing nanoparticles having the diameters and intervals in the above- Lt; RTI ID = 0.0 > embryonic < / RTI > stem cells without supporting cells.

Preferably, the embryonic stem cell may be maintained at an expression level of Oct4 at a level of more than 90%.

A cell culture container comprising nano pillars uniformly arranged in a uniformly decreasing spacing of nano pillars having uniformly increasing diameters of the present invention provides various surface morphologies in one culture container Therefore, it is possible to simultaneously monitor the effect of changes in the surface morphology on the cell characteristics by the single culture performed under the same conditions, and the optimum surface morphology suitable for cell culture can be selected using the same. The method of culturing the selected human embryonic stem cells through the screening can provide a method capable of proliferating while maintaining the stem cell function without coating the costly extracellular matrix or using the supporting cells.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram schematically illustrating a method for producing a gradient nanopattern (G-Np) according to the present invention. (A) schematically shows a method for producing a nanoporous AAO template having an increased pore diameter, anodizing the template with phosphoric acid and gradually increasing the pore-widening time to increase the diameter gradient of the pore . (B) shows the relationship between pore expansion time and pore diameter. The pore diameter increased linearly with increasing pore expansion time. The inserted SEM image shows the pore diameter at the indicated pore extension time (scale bar = 1 Ym). (C) schematically shows a process of forming a nanopillar on a PS surface by a thermal nano-imprint method using an AAO template having a pore gradient. (D) is a nano-column diameter gradient array on G-Np, indicating that the increase in the diameter of the nanoparticles decreases the spacing at the same time. The inserted SEM image shows the specific spatial arrangement of nanopillars in the indicated area of G-Np (scale bar = 1 Ym).
FIG. 2 is a graph showing quantitatively the pore size of the nanoporous AAO used in the production of G-Np according to the present invention with respect to the pore expansion time. (A) is a SEM image of AAO according to pore expansion time. (B) shows the change in the diameter of the pores according to the pore expansion time, and shows that the diameter of the pores linearly increases with the pore expansion time.
3 is an SEM image of G-Np coated with gelatin.
Figure 4 shows the growth characteristics and Oct4 expression of single hESCs cultured on an unstructured control surface (control) and G-Np according to the present invention. (A) is an optical microscope image of a hESC cultured on an unstructured control surface, showing morphological heterogeneity on the first day after plating, and a single cell of a wide spread cell on the fourth day A layer was observed (scale bar = 200 Ym). (B) shows an optical microscope image of hESCs cultured on G-Np, maintaining a spherical shape with no signs of diffusion on the first day after plating, and organized into a typical dense population. (C) shows the result of immunocytochemical analysis. It was confirmed that the colonies cultured on G-Np showed a higher level of Oct4 expression than the cells cultured on the control surface. (D) control and Oct-4 expression in colonies grown on G-Np. The hESCs cultured on the control panel showed significantly reduced levels of Oct4 expression (~ 58%) compared to those appearing on different regions of G-Np (low; ~ 92%, mid; ~ 78% and high; ~ 82% Respectively.
Figure 5 shows E-cadherin expression of single hESCs cultured on an unstructured control surface (Control) and G-Np according to the present invention.
FIG. 6 is a graph showing hESCs diffusion, cytoskeletal white matter and E-cadherin expression. (A) shows the result of phalloidin staining and shows hESCs diffusion on the contrast surface visually. On the other hand, it was confirmed that the hESCs cultured on G-Np were maintained as colonies. (B) shows the average distance from the center of the colonies to the nucleus of each cell, quantitatively showing the cell diffusion in hESC colonies. The hESCs on the control panel formed a broad colony and spread to ~ 220 μm. The extent of diffusion of hESCs on G-Np varied with the area of the pattern. The colonies observed in the low region (~ 100 μm) exhibited minimal diffusion (P ≤ 0.001) compared to those observed in the medium (~ 120 μm) or high region (~ 130 μm). (C) to (F) show the results of immunocytochemical analysis on the face of the control and the different regions on G-Np, respectively. As shown in (C), the expression of E-cadherin was decreased in the hESC colonies cultured on the control surface, and E-cadherin was isolated from paloidin staining according to hESC diffusion. As shown in (D) to (F), it was confirmed that E-cadherin was overexpressed at the cell-cell boundary in the colon in all three regions of G-Np. It can be inferred that E-cadherin expression is mediated by actin cytoskeletal binding, which is the expression of E-cadherin mediating cell-cell adhesion from paloidine staining and superimposition.
Figure 7 shows focal adhesion formation and membrane protrusions of hESCs colonies cultured on the control surface and G-Np. (A) is a schematic representation of focal adhesion assembly for integrin and ECM interactions. The introduction of a variety of local attachment proteins leads to actin stress fibers ) Is anchored. (B) shows the results of vinculin and paloidin staining. The remarkable expression of vinculin and palodidin indicates anchorage of actin stress fibers in lesion attachment, indicating that colonization has already occurred on the control plane And it is diffused. In the tip of filopodia staining on G-Np, the expression of vacanculin was observed exclusively at the end of hESC colonies (white arrow). While there were a large number of thought families in the hESCs colonies observed in the lower regions, colonies observed in other regions indicated that they formed lamellipodia and thought families. (C) are SEM images of hESCs colonies cultured on three different regions of G-Np. At the hESC end, lamellipodial extensions were identified in the middle and high regions (white arrows). In the low region, proliferation of the Thrush group at the distal end of the spherical hESC colonies and limited protrusion of Lamellipodia were observed. The hESC colonies also remained spherical in the middle and high areas, but flat and broad protrusions of lamellipodia were prominent (the black and white arrows indicate the Spans and Lamellipodia, respectively). (D) is a more enlarged image, confirming that the protrusions of filamentous fungi and lamellipodia are specifically observed in the upper part of the nano pillars (F; filopodia and L; lamellipodia; ).

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Nano-gradient surface ( gradient nanopattern ; G- Np )

Ultrapure aluminum plates (99.999%, Goodfellow, UK) were electrochemically cleaned with perchloric acid (absolute ethanol = 1: 4) at 7 ° C using a 20V voltage. The washed aluminum plate was anodized with a phosphoric acid solution (DI water: methanol: phosphoric acid = 59: 40: 1) at a voltage of 193 V for 12 hours at 0 ° C., and a chromic acid solution (9 g chromic acid and 20.3 ml phosphoric acid in 500 ml DI water). The anodic aluminum oxide (AAO) containing the well-aligned pores prepared above was again subjected to an anodic oxidation treatment under the same conditions for 4 hours, and a phosphoric acid solution was applied using a tensiometer (operating speed: 1.0 mm / min) , And the intensity was controlled while gradually increasing the dipping time. The AAO template containing the controlled gradient pores was coated with a self-assembled monolayer of heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane (HDFS). The surface of the AAO was first hydroxylated with piranha solution (35% H ₂ SO ₄ : H ₂ O ₂ = 7: 3, vol / vol) and washed with a 3 mM aqueous-free HDFS n-hexane solution So that an HDFS self-assembled monolayer was formed on the surface. A polystyrene (PS) nano-gradient surface (G) was formed by thermally imprinting an AAO template containing pores having a gradient diameter of nanometer unit using a nanoimprinting device, NANOSIS ^TM 610 (Nano & Device, Korea) -Np). A PS polymer sheet of 700 mm ² (35 mm × 20 mm) size was heated at 130 ° C. for 10 minutes and compressed at 3 bar for 100 seconds. After lowering the temperature to room temperature, the AAO template was separated from the PS sheet.

For the G-Np preparation, anionic aluminum oxide was prepared containing a regular array of pores with a center-centered pitch of 440 nm by two-step anodization in phosphoric acid. AAO having a pore gradient of nanometer size was obtained by slowly increasing the immersion time from 0 to 350 minutes in the phosphoric acid solution for pore expansion at 30 DEG C (FIG. 1A). In AAO, the pore diameter increased linearly from 100 nm to 320 nm as the immersion time increased (FIG. 1B and FIG. 2). The isotropic etch rate was 0.65 nm / min, which was similar to the previously reported values (Fig. 1B). PS surface patterning with varying diameter nano pillars was performed by thermal nanoimprinting of AAO templates with nanoporous gradients (Figure 1C). PS, which is the most widely used material in commercial cell culture dishes, was chosen as the bottom material. A continuous gradient of the nanopillar diameter was determined (diameter D; 120-360 nm, interval S; 320-80 nm, FIG. 1D) at a constant pitch length reducing intervening spacing from the SEM image. Also, the diameter of the column coincided with the diameter of the corresponding pore of the mold. (Low, D = 120-170 nm), "medium" (mid, D = 170-290 nm) and "high" (low) depending on the diameter range of the nanoparticles for easy identification of other regions of G- high, D = 290-360 nm).

Example  2: Cell culture

H9 hESCs (Wicell, WI) were cultured by a known method as previously reported. Specifically, small clumps of human embryonic stem cells (hESC) colonies were excised with a glass Pasteur pipette and stained with mytomycin-C (Sigma-Aldrich, St. Louis, Mo.) ), 20% (v / v) serum replacement, 100 mM β-mercaptoethanol, 1 mM L-glutamine (L- F12 (Gibco BRL, Gaithersburg, MD) medium containing 1% (v / v) non-essential amino acid and 4 ng / ml bFGF (Invitrogen, Grand Island, NY) Respectively. Cells were cultured for 6 days while exchanging the medium every day. In order to dissociate into single cells, hESCs were incubated at 36 ° C for 1 hour with the addition of 10 μM rho associated protein kinase (ROCK) inhibitor Y-27632 (Tocris, MO). The hESC colonies were then treated with Dispase (Gibco) for 5 minutes at 36 ° C. The lifted hESC colonies were washed with PBS (Sigma-Aldrich) and incubated with TrypLE ^™ -select (Invitrogen) for 3 minutes at room temperature. The pellet was gently pipetted to physically separate the clot and centrifuged at 900 rpm for 2 minutes. The pellet was resuspended in hESC culture medium and the plug was filtered through a 5 ml polystyrene round-bottom tube with cell-strainer cap (BD Falcon) containing cell filters to remove any remaining cell aggregates. Cells were counted using a hemocytometer and an unstructured control surface containing hESC medium containing 10 ng / ml bFGF and 10 μM Y-27632 and the G- Lt; ² > cells / cm < ² > on Np. To facilitate attachment of single hESCs, the control panel and G-Np were coated with 0.1% gelatin (Invitrogen) for 20 min at room temperature before dispensing the cells. The hESCs were cultured for 4 to 5 days while exchanging the medium on a daily basis.

To determine if the increasing diameter nanopillar array affects hESC colony formation, G-Np was first coated with 0.1% gelatin. It was confirmed by SEM that the nano-pillar shape protruding from the gelatin was uniformly coated over the pattern without selecting it (Fig. 3). The hESC colonies were then separated into single cells using enzymes and cultured on G-Np for 4 days to compare with the growth characteristics of cells cultured on unstructured control surfaces. On the control panel, most of the adherent single hESCs were flattened and lengthened to show fibroblast-like morphology after one day of plating (Fig. 4A). Subsequently, the cells migrated toward the neighboring cells while dividing cells instead of forming colonies, and the daughter cells spread out from each other in an isotropic manner and formed a single layer spread widely on the fourth day after plating (Fig. 4A ). In contrast, a single hESC plated on G-Np exhibited a dense spherical morphology with minimal expansion of cytoplasm (Fig. 4B). Subsequent adherent single hESCs migrated toward neighboring cells, forming aggregates and forming individual colonies at the onset of cell division (FIG. 4B). The colonies continued to proliferate within the colonies without any indication of cell proliferation (Fig. 4B). Four days after plating, the colonies showed dense, well-defined boundaries, which are available as a morphological criterion for pre-differentiating hESC colonies (Fig. 4B).

Example  3: SEM Imaging

The hESC colonies grown on G-Np for 3 days were washed twice with PBS and fixed with 4% para-formaldehyde for 4 hours at room temperature. The prefixed hESC colonies were fixed with 1% osmium tetroxide for 2 hours, and ethanol was dehydrated by changing the concentration from 50% to 100%. The dehydrated hESC colonies were washed 3 times with t-butanol, frozen at -20 ° C and lyophilized for 1 day. The completely freeze-dried hESC colonies were plated with platinum for 5 minutes and observed with SEM (scanning electron microscope, FE-SEM, JSM6701, JEOL, Japan). All images were acquired with a 40 ° slope.

The present inventors have hypothesized that the pandemic complexes abundant around the hESC colonies are due to the specific nanoparticle diameter range in the low region (D = 120-170 nm), which provides a disadvantageous nanograid for the elongation of the filament family and lamellipodia . To test this hypothesis, we analyzed the formation of filarians and lamellipodia in hESC cultured on G-Np by SEM. In the low region (D = 120-170 nm), the hESC colonies showed spherical shapes with fine circumferential outgrowth of the species and limited protrusion of Lamellipodia. Colonies found in the middle (D = 170-290 nm) and high (D = 290-360 nm) regions are also flattened, including small filopodia outgrowth around the colonies, The overhang of the large Lamellipodia indicates that the colonization has spread (Figure 7C, white arrow; Ideological group, black arrow; Lamellipodia). Membrane protrusion structures were observed specifically at the top of the nanopillar array (Fig. 7D), indicating that the nanopillar is an attachment region for the caspase complex and lamellipodia protrusion. From this, nanopillar arrays with diameters in the range of 120 to 170 nm provide insufficient regions for complex clustering of the lesions and thus hESC lamellipodia protrusion can be restricted to maintain dense colony integrity.

Example  4: Immunocytochemical analysis Immunocytochemistry assay )

Cells cultured on the control panel and G-Np for 4 days were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 minutes. The samples were then treated in turn with permeabilizing (0.03% Triton-X in PBS for 10 minutes) and blocking solution (5% goat serum in permeabilizing solution for 30 minutes) at room temperature. The cells were stained with Oct4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, 1: 1000) and E-cadherin (BD, 1: 200), actin cytoskeleton marker pyrophylidin incubated at 4 째 C overnight in a blocking solution containing the primary antibody against Pallidol, Invitrogen, 1: 400) and the local attachment marker vinculin (Sigma-aldrich, 1: 100). Cells reacted with the primary antibody were washed three times with PBS and incubated with Alexa Flour 488- or 594-conjugated secondary antibody (Invitrogen) at room temperature for 2 hours and washed three times with PBS for 5 minutes each. After fluorescent staining with DAPI (6-diamidino-2-phenylindole, Sigma, 1: 1000) in PBS for 3 minutes at room temperature, fluorescent images were obtained using a fluorescence microscope (Nikon TE2000-U, Nikon, Chiyoda-ku, Japan) .

To confirm whether hESC diffusion is essentially involved in maintenance of the ability to differentiate, we performed immunocytochemical assays using antibodies against Oct4, a well-known pre-differentiation marker. The control panel not only induced isotropic spread of single hESCs but also markedly reduced Oct4 expression (~ 58%). On the other hand, hESCs organized with well-defined colonies on G-Np expressed Oct4, E-cadherin and other fully differentiable markers at much higher rates (Fig. 4C and Fig. 5). Oct4 expression levels showed marked differences between the hESC colonies observed in other regions of G-Np. The colonies observed in the lower region of G-Np (D = 120-170 nm) expressed Oct4 at the highest level (91%) followed by the higher region (85%, D = 290-360 nm) (79%, D = 170-290 nm) (p? 0.05, Fig. 2D- > 4). 1) G-Np provides a nano-topical solution for systematizing a single hESC into a well-organized dense colony, 2) collapse of dense colony integrity is detrimental to the hESC pre-differentiation ability under supported cell free conditions, and 3) We confirmed that the specific nanoparticle diameter range (D = 120-170 nm) is the optimal nanostructure for Oct4 expression.

The fact that the hESC colonies observed in the lower regions (D = 120-170 nm) also indicate higher Oct4 expression levels compared to those observed in other regions suggests that tight cellular association and hESC It emphasizes the close relationship between maintenance of differentiation. Within the hESC colonies, tight cell-cell adhesion is mediated by E-cadherin in the cadherin superfamily, whose expression is closely related to hESC survival and maintenance of pre-differentiation potential. We identified strong expression of E-cadherin at the cell-cell interface within the colonies observed on G-Np, and the E-cadherin overlapped with Paloidin staining (Figures 6D-6F). Indicating that the adhesion of E-cadherin is mediated by actin cytoskeletal binding through alpha-and beta-catenin. On the other hand, E-cadherin, which mediates cell binding, decreased significantly when single hESCs cultured on the control surface were diffused from each other. Which was confirmed by an E-cadherin expression patch at the cell boundary (Fig. 6A, arrow). In addition, E-cadherin expression isolated from paloidin-staining demonstrates that actin cytoskeletal reorganization during hESC diffusion destroys the binding between E-cadherin and actin cytoskeleton, thereby eliminating intercellular adhesion mediated by E- cadherin (Fig. 6A, arrow). As shown in FIG. 4, 1) hESC diffusion has mechanosensitivity and increases as a function of nanoparticle diameter, and 2) a specific range of nanopillar diameters (D = 120-170 nm) 3) support for E-cadherin mediates tight cell binding, which is essential for hESC pre-differentiation potential, while limited hESC spreading on G-Np supports hESC pre-differentiation.

Next, we observed how hESCs responded to increased nanoparticle diameters during colonization. Topological sensing of adherent cells is accomplished via surface membrane integrins at the terminal ends. First, anchorage-dependent cells formed nascent contact adhesions (focal complexes) that protrude the fasciculus and induce lamellipodia kidneys to sense local phase. Clustering and increased density of complexes form more stable attachment sites (focal adhesions) and actin-stress fibers (actin filament bundles) are fixed on the attachment sites to maximize cell / surface boundary area, resulting in additional membrane flattening (Fig. 7A). During cell diffusion, lesion adhesion and actin cytoskeletal organization are largely influenced by the surface topography of the basal matrix. When the spacing between nanostructures exceeds 70 nm, the diffusion of the fibroblasts is limited, while the nanotubes with 100 nm lateral spacing inhibit the formation of surface film protruding structures (e.g., filaments and lamellipodia) It is known to limit cell proliferation. Enzymatically isolated hESCs were cultured on unstructured control plates and G-Np for 4 days in order to examine how nanoparticles affects lesion attachment and actin cytoskeleton organization during the hESC colony formation process. Immunocytochemical analysis revealed that the hESCs cultured as shown by punctate vaculin expression at the ends of the paloidine-positive actin cytoskeleton on the control panel formed a stable lesion attachment at the end of the diffusing hESC (Fig. 7B, White dotted circle). Significant staining of paloides shows the organization of the actin cytoskeleton into actin stress fibers, which is fixed to lesion attachment and induces membrane diffusion. This indicated that individual hESCs on the control panel diffuse instead of organizing into dense colonies. When hESCs were cultured on G-Np, considerable changes in lesion-associated assembly and actin cytoskeletal organization were observed (FIG. 7B). When the cells were tightly linked to each other, no stained viral coolin expression was observed in the colonies (Fig. 4B). This may account for the limited hESC colony spread observed on G-Np. Viculline expression at the periphery of the hESC colonization occurred at the terminal end of the caspase, suggesting the formation of nascent focal complexes instead of stable lesion attachment (Fig. 7B, white arrow). More pandemic complexes were observed around the hESC colonies cultured in the lower regions (D = 120-170 nm) as compared to the other regions of G-Np (Fig. 7B, white arrow).

Example  5: Quantitative analysis ( Quantitative 분석 )

Ten representative phalloidin-labeled colonies were randomly selected from three different regions on the control panel and G-Np to analyze the extent of hESC colonization. The nuclear distance of each cell was measured from the center of the colon using ImageJ software (http://rsb.info.nih.gov/ij/). Percentage of expression of Oct4 was measured at the location where 10 identical representative colonies were randomly selected from the control and G-Np using the same software. Data were expressed as mean ± standard error of the mean (SEM) and one-way analysis of variance (ANOVA) was performed using SPSS software (Chicago, IL) , P < 0.05 was considered statistically significant.

G-Np organizes a single hESC into tight colonies by limiting diffusion, and we further analyzed the effect of varing nanoparticle diameters on hESC diffusion during colonization. Immunocytochemistry using the cytoskeleton marker, paloidine, visualized hESCs in three different G-Np regions exhibiting individual hESCs and limited diffusion by diffusion on the control panel (Fig. 6A). To determine the degree of diffusion of hESCs, the mean distance of each cell nucleus from the center of the colonies was measured. A single hESC spread on the control surface by ~ 200 nm to form a broadly spread cell monolayer (Figure 6B). In contrast, the hESCs cultured on G-Np were significantly less diffuse, with colonies observed especially in the lower regions of G-Np showing the most compact colonies compared to those observed in other regions (Figure 6B) .

Example  6: Real Time Video Imaging ( live video imaging )

Growth of the hESCs cultured on the control panel and G-Np was recorded by taking a time-lapse image every two minutes for two days. Images were taken using a Nikon ECLIPSE TS 100 microscope and placed on a Tokai Hit Incubation System (Tokai Hit Co., Ltd., Shizuoka-ken, Japan) for microscopic observation.

Claims (12)

A method for screening a surface structure suitable for embryonic stem cell culture, comprising culturing cells in a cell culture vessel containing a gradient nanopillar array having a constant diameter or spacing.
The method according to claim 1,
Further comprising the step of analyzing the characteristics of the cultured embryonic stem cells harvested from the region including the nanoparticles of different diameters and spacing of the gradient nanopillar array with constant diameter or spacing varying.
3. The method of claim 2,
Wherein the step of analyzing the characteristics of the cultured embryonic stem cells is performed by measuring the expression of a pre-multipoint marker.
The method of claim 3,
Wherein the pre-multipoint marker is Oct4.
3. The method of claim 2,
Wherein the step of analyzing the characteristics of the cultured embryonic stem cells is performed by further measuring the expression of a cell adhesion marker, a cytoskeletal marker or a lesion attachment marker.
6. The method of claim 5,
Wherein the cell adhesion marker is E-cadherin, the cytoskeletal marker is phalloidin, and the lesion attachment marker is vinculin.
The method according to claim 1,
And screening for a condition capable of culturing embryonic stem cells while maintaining stem cell morphology without supporting cells.
A cell culture container comprising nano pillars uniformly arranged at intervals such that the nano pillars having a constantly increasing diameter used in the screening method according to claim 1 are uniformly reduced.
9. The method of claim 8,
Wherein the nanopillars have a diameter of 100 nm to 400 nm and are arranged at an interval of 350 nm to 50 nm.
A cell culture vessel for maintaining stem cell function comprising nano pillars having nano pillars having a diameter of 120 nm to 170 nm uniformly arranged at intervals of 320 nm to 250 nm.
And culturing the cells without supporting cells in a cell culture container containing nanoparticles uniformly arranged at intervals of 320 nm to 250 nm with diameters of 120 nm to 170 nm. A method for culturing embryonic stem cells.
12. The method of claim 11,
Wherein said embryonic stem cells are maintained at an expression level of Oct4 at a level of greater than 90%.

Images