JP2015177755A - Groove structure which expresses cell exclusion properties - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable production of a highly sensitive cell chip which is stable for a long period of time, a cell sheet with high safety, and the like having a high necessity in foundation and clinical medicine, implant, and regenerative medicine field by locating a surface expressing a stable and strong cell expulsion properties adjacent to around a cell affinity surface.SOLUTION: The invention provides a substrate having on its surface a geometric structure consisting of V-groove structure of single or plural sequences, the substrate stably inhibiting adhesion, proliferation, expansion, and transfer of cells for a long period of time. By locating, adjacent to around a cell affinity region, a region which expresses cell exclusion properties due to the geometric structure, a cell pattern formed on the substrate is made to flexibly correspond to a target, an application, and the like to enable production of a cell chip, a cell sheet, and the like in the foundation and clinical medicine, implant, and regenerative medicine field.

Description

  The present invention relates to a groove structure processed on the surface of a cell chip, a cell culture substrate, or the like that stably expresses cell exclusion over a long period of time by suppressing cell adhesion, proliferation, extension / migration. It is. By arranging the cell exclusion area consisting of the above-mentioned single or multiple rows of V-shaped groove structure around the smooth cytophilic area on the substrate, cell patterning and tissue formation using cells etc. Contributes to cell engineering or medical applications.

As represented by DNA chips and protein chips, a technique for immobilizing biomolecules on a micro area of μm scale on the chip surface and performing high-throughput analysis has recently been developed and frequently used. Furthermore, recently, a cell chip in which cells are arranged in an array on a chip has been developed, and high-throughput functional analysis of genes and proteins at the cell level has begun to be applied to the field of medical diagnosis.
In order to perform high-throughput analysis of cell function evaluation, it is necessary to culture cells by attaching them to the chip surface in the same manner as other biochips. So far, several culture techniques have been reported in which cells are selectively adhered and arranged only on a minute part on a substrate. Once such stable sequence control technology for cultured cells derived from humans and animals is established, it will be possible to industrially apply to cell chips having sensing functions, bioreactors for the production of biologically useful substances, etc. In addition, application to culture substrates for stem cell culture such as iPS cells is expected to be applied to advanced medical fields such as regenerative medicine and hybrid artificial organs.

As a method for controlling the arrangement of cultured cells, conventionally, using a base material on which surfaces having different ease of adhesion to cells form a pattern on the same plane, cells are cultured on this surface, A method is used in which cells are arranged by adhering only to the surface that has been processed so that the cells adhere.
For example, Patent Document 1 attempts to arrange cultured cells on a surface obtained by micropatterning a cell non-adhesive or cell adhesive polymer by photolithography. Typical examples of the cell adhesion polymer include cell adhesion proteins such as collagen and fibronectin, and examples of the cell non-adhesion polymer include hydrophilic polymers having no charge such as polyvinyl alcohol and polyethylene glycol.
Patent Document 2 discloses a cell culture substrate patterned with a substance such as collagen that affects the cell adhesion rate and morphology, and a method for producing the substrate by photolithography. By culturing cells on such a substrate, many cells are adhered to the surface on which collagen or the like is patterned, thereby realizing cell patterning.
In Patent Document 3, a non-cell-adhesive surface is prepared by applying a cross-linked albumin treated with a cross-linking agent made of a polyepoxy compound onto a substrate, and cell-adherent cells such as collagen and fibronectin are formed on a part thereof. Micropatterning of cells is possible by arranging the outer matrix by inkjet printing technology.
Patent Document 4 reports a cell adhesion control material characterized in that an irradiation site becomes non-cell-adhesive by irradiating an ion beam to gelatin or collagen coated on a substrate.
However, these currently known cell array methods use a culture substrate in which proteinous cell adhesion factors or the like are fixed on the substrate surface or the surface is hydrophobized / hydrophilicized by a chemical method. However, due to surface stability and uniformity problems, there is an essential problem that cell adhesiveness changes during the culture period, and long-term cell culture and cell adhesion / non-adhesion are completely eliminated. Sorting and patterning is difficult, and there are significant limitations in application to cell patterning, tissue formation in and outside the body, and the like. Furthermore, the modification of adhesion by ion implantation as in Patent Document 4 is accompanied by a disadvantage that a large and expensive ion implantation apparatus is required.

  In order to solve these problems, a surface that exhibits stable and robust cell exclusion so that the cell pattern can be maintained for a long time without using non-cell-adhesive chemicals that are easily detached from the substrate surface during culture. The creation of

Japanese Patent Publication No. 7-51061 Japanese Patent Laid-Open No. 5-176653 JP 2012-187072 A JP-A-7-108060 JP 2011-155865 A Japanese Patent No. 4934360

The present invention provides a stable and robust surface-excluded surface adjacent to the periphery of the cytophilic surface, thereby providing a long-term stable high-requirement that is highly necessary in the basic and clinical medicine, implant, and regenerative medicine fields. The object is to enable the production of sensitive cell chips and highly safe cell sheets.
In conventional cell adhesion or non-adhesion region micropattern fabrication methods, non-cell-adhesive chemicals are used in photomask processing and stamping methods, and polyethylene glycol (PEG), etc., forms cell non-adhesion regions exclusively. The PEG-modified region and the PEG-unmodified region are used as a cell non-adhesion region and a cell adhesion region, respectively.
However, the conventional cell array method using the chemical properties of the surface of the substrate has a problem that the adhesiveness and the like change during the culture period due to surface stability and uniformity. In principle, it is difficult to perform patterning by completely sorting cell adhesion and non-adhesion of cells for a certain period of time, and there are significant limitations in application to cell patterning, tissue formation, and the like. In order to solve this problem, the creation of a stable and robust cell-exclusion surface that can maintain the cell pattern for a long time without using non-cell-adhesive chemicals that are easily detached from the substrate surface during culture is strong. It has been demanded. If chemical substances are not used, the chemical substances will not be detached from the culture medium, so there will be little effect on the cultured cells, the cell chip will have high analysis accuracy, and if used for regenerative medicine, Safety can be improved.

  The present invention provides a cell culture substrate comprising a single or multiple rows of V-groove structures, and a surface that expresses a stable and robust cell exclusion property adjacent to the periphery of the cell affinity surface. Is a solution.

The present inventors have conducted extensive research in view of the problems associated with conventional cell culture methods using non-cell-adhesive chemicals, and as a result, have obtained a cell-scale geometric structure (V-groove structure) formed on the substrate surface. By initial adjustment of the depression angle of the V-groove structure and the radius of curvature of the groove bottom, the initial adhesion of the cells seeded in suspension to the V-groove structure is significantly suppressed. In addition, a small number of cells adhered to the V-shaped groove structure are confined in a minute space within the groove structure, thereby changing to a cell shape different from that on the plane, and as a result, cell proliferation and extension are suppressed. As a result, the present invention has been completed.
That is, the present invention relates to a cell for culturing cells used in cell chips, implants, and regenerative medicine, and a cell having a V-shaped groove structure having an appropriate shape and size adjacent to the periphery of the cell affinity region on the surface of the substrate. By placing an exclusion surface, the culturing of cells seeded on the substrate can be controlled without using cell-exclusion chemicals such as those used in the past. Providing a substrate.

A conceptual diagram of a cell culture substrate provided with a V-shaped groove structure according to the present invention is shown in FIG. The substrate of the present invention is a cell-affinity region (B region in the figure) consisting of a smooth surface that does not inhibit cell adhesion, proliferation, extension / migration, and cell adhesion / proliferation provided adjacent to the periphery. The cell exclusion region (A, An-1, An region in the figure) in which the V-shaped groove structure that inhibits extension / movement is processed is used as a structural unit.
By arranging the V-shaped groove structure in a plurality of rows according to the cell mobility (upper right figure), it is possible to further suppress the cell migration between the cell adhesion planes than in a single row.
Furthermore, by changing the cell adhesion plane from a convex island structure to a concave hole structure (lower right figure), cell mobility is further suppressed.

The V-shaped groove structure used in the present invention is a V-shaped groove having a depth and a width that allow spherical cells suspended in a culture solution or the like to enter completely, and an angle formed by the groove wall surface is 53 degrees or less. It is characterized by being.
The groove having such a depth and width can prevent cells from directly growing on the substrate surface across the groove, and the groove wall surface having such a depression angle can prevent the groove from growing. The cells that enter can be prevented from normal growth / proliferation of the cells by being placed in a narrow space between the groove wall surfaces, which is different from the cell affinity region.
From such a viewpoint, the groove structure of the present invention preferably has a radius of curvature of the bottom surface of the groove of 20 μm or less and smaller than the radius of curvature of the cell.
From this point of view, the width of the V-shaped groove of the present invention is a size that allows the cells to be completely accommodated in the V-shaped groove. Among the human and animal-derived eukaryotic cells having a size of about 5 μm, the size is preferably about 15 μm or more.

The present invention enables cell patterning, networking, tissue construction, etc. required in the field of cell chips, implants, and regenerative medicine by controlling cell dynamics using the above means. It is.
In addition, as a substrate material, substrates of various materials used for normal cell chips, implants, and regenerative medical materials such as glass, crystal, metal, and resin can be used. In addition, as a processing method of the V-shaped groove structure on the surface of the substrate, any known method suitable for the material of the substrate such as cutting, focused ion beam, sand blasting, injection molding, contact imprint, lithography, etc. may be used. It is possible to select.

Several techniques for providing a geometric structure on a cell culture substrate have been known before the present invention.
For example, Patent Document 5 describes providing a plurality of polygonal column-shaped structures produced by a lithography method as an inhibition region for inhibiting cell migration and adhesion, as in the present invention. The length of one side of the top surface of the structure is 3 μm or more and 20 μm or less, and the interval between the structures is 0.5 μm or more and 1.5 μm or less.
In Patent Document 5, the interval between the structures is made smaller than the size of the cells, thereby preventing the cells from adhering to the structure and moving beyond the region of the structure.
Moreover, in patent document 6, the cell support body comprised from the environmentally responsive polymer for forming a cell sheet which consists of the convex part which has a top | upper surface, and the recessed part formed between this convex part is reported. The support is made non-adherent to the cell sheet that grows and proliferates across the recess by setting the width of the opening of the recess to 0.1 to 10 μm, which is a size in which cells to be cultured cannot infiltrate. By making it sex, the obtained cell sheet is easily peeled off from the support.
In contrast, according to the present invention, by providing a V-shaped groove having a width that allows cells to infiltrate, it is possible to prevent cells from directly proliferating across the V-shaped groove, and cells that have entered the V-shaped groove are It has been found that normal growth is prevented and growth along the V-shaped groove is also prevented, and the adhesion and proliferation of cells by a structure that is simpler and easier to produce than the method of Patent Document 5. , To achieve the inhibition of extension and movement.

That is, this application provides the following invention.
<1> A V-shaped groove that inhibits cell adhesion, proliferation, extension, and migration, and has a depth and width that allow a spherical cell suspended in a culture solution or the like to completely enter. A cell-excluded groove structure characterized by a depression angle of 53 degrees or less.
<2> The cell-excluded groove structure according to <1>, wherein the curvature radius of the groove bottom is 20 μm or less.
<3> A cell culture substrate on which the cell-exclusion groove structure according to <1> or <2> is formed.
<4> The cell culture substrate according to <3>, wherein the cell-exclusion groove structure is formed around the cell affinity region.
<5> The cell culture substrate according to <3> or <4>, wherein the cell-exclusion groove structure is provided in a single row or in a plurality of rows.
<6> The cell culture substrate according to any one of <3> to <5>, wherein the cell affinity region is a smooth plane.
<7> The cell culture substrate according to any one of <3> to <6>, wherein the cell affinity region is surface-modified with a cell adhesive protein.
<8> The cell culture substrate according to any one of <3> to <7>, wherein the cell culture substrate is used as a cell chip, an implant material, or a cell sheet culture substrate.
<9> The cell-excluded groove structure according to <1> or <2>, wherein the groove is processed by any one of cutting, focused ion beam, sandblasting, injection molding, contact imprint, and lithography. Manufacturing method.

According to the present invention, cells normally adhere, proliferate, and differentiate in a planar cytophilic region (island structure or hole structure) that is not subjected to V-groove processing. On the other hand, in the cell exclusion region that has been subjected to V-shaped groove processing, cell adhesion, proliferation, extension, and movement are remarkably suppressed by the minute V-shaped space. As a result, the cell adhesion region and the non-adhesion region can be clearly separated on the surface of the substrate.
A cell adhesion pattern can be freely set on a substrate by patterning and arranging a cell affinity region having a cell exclusion region with a V-shaped groove formed around it. Thereby, cell patterning, networking, tissue construction, etc. required in the field of cell chips, implants, and regenerative medicine are possible.
In the cell chip to which the present invention is applied, not only analysis but also introduction of areas that do not mix with each other independently by mixing drugs and genes into different cell adhesion substances in the cell affinity region. This makes it easier to obtain desired clonal cells. Therefore, it becomes possible to screen and evaluate drugs and genes based on various cell phenotypes (cell motility, differentiation, cell death, cell proliferation, etc.).
According to the method of the present invention, since the micropattern of the cell adhesion region can be formed easily and at a lower cost than the conventional photomask processing and stamp method, the culture substrate can be made into a microarray and used. This makes it possible to evaluate drugs and genes quickly, simply, and inexpensively. Thus, the present invention provides a new drug development, gene analysis tool, and culture substrate for regenerative medicine.
As described above, the culture substrate of the present invention can surely control the dynamics such as local cell adhesion, proliferation, extension, and movement, and also has a V-groove structure formed by directly surface processing the substrate material. Since this is controlled by physical structure such as the shape and width and depth of the cells, there is no detachment or denaturation from the surface as in the case of using chemical substances, and cells in the cell exclusion site Exclusion is maintained during the culturing period, and long-term culturing and patterning of living cells can be easily realized, so that formation of living cell networks, tissue formation, and the like are possible. In addition, since the culture substrate of the present invention does not reach the cell as the chemical substance acts as in the case of using a chemical substance, when used as a cell chip, it becomes possible to perform an accurate chip analysis of the test sample, Even when used as an implant or a material for regenerative medicine, there is a merit that there is no contamination of chemical substances and a safer treatment is possible.

The conceptual diagram of the groove | channel structure by this invention. A glass substrate (A-1: bird's-eye view with an electron microscope, A-2: cross-sectional image with a confocal laser microscope) and a U-grooved glass substrate (B-1) with V-groove processing on the surface by sandblasting : Bird's eye view with electron microscope, B-2: Cross-sectional image with confocal laser microscope). Staining images of nuclei of adherent cells on day 1 after seeding (A-1, B-1: cell affinity region, A-2, B-2: V-shaped groove, U-shaped groove). White yajiri: DAPI-stained cell nucleus. Stained images of nuclei of proliferated cells on day 14 after seeding (A-1, B-1: cytophilic region, A-2, B-2: V-shaped groove, U-shaped groove). White yajiri: DAPI-stained cell nucleus. Stained images of differentiated cells of alkaline phosphatase 14 days after seeding (A-1, B-1: cell affinity region, A-2, B-2: V-shaped groove, U-shaped groove). White tapping: alkaline phosphatase positive cells, black tapping: hematoxylin stained cells.

  The present invention has realized the purpose of controlling the fixed and excluded areas of cells in a substrate, regardless of the type of cells, the substrate, etc., by means of an excellent substrate surface geometry that is simple and highly accurate. The substrate used here is not limited as long as it is a solid material having a smooth surface. Examples thereof include, but are not limited to, glass, quartz, and plastic depending on the purpose of use such as cell chips, implants, and regenerative medical materials. Further, the surface shape may be either a flat surface or a curved surface depending on the method of processing the geometric structure, and is not limited to a flat plate.

  The “cell” as a target in the present invention is typically an adherent cell line or a cancer cell, a primary cell isolated from normal or cancerous tissue of a mammal, or an artificial cell such as an ES cell or an iPS cell. Including engineered cells. Suspended cells such as blood cells that do not exhibit substrate adhesion are excluded from the subject of the present invention due to their nature.

In the present invention, the process of arranging cells on a substrate means a step of adhering specific cells based on a desired arrangement pattern. That is, preparation of a cell chip in which spots of a plurality of cells are aligned and fixed in a certain region, formation of a regenerated tissue induced from proliferation and differentiation from stem cells such as iPS cells, or in vivo implantation. The engraftment of specific cells on the inserted implant is included. In order to successfully place cells on the designed pattern, not only cell adhesion to the cell affinity region but also cell adhesion, proliferation, extension / migration to other regions can be suppressed.
In order to ensure cell adhesion to the cell affinity region, a surface coating such as a cell adhesion protein such as collagen or fibronectin is effective.
Furthermore, these extracellular matrices can be used for gene transfer via cell adhesion surfaces to cells cultured in the cell affinity region by mixing nucleic acid substances such as DNA and RNA.
For cell seeding in the cell affinity region, it is possible to apply a conventional technique in which a cell-containing solution is brought into contact with the entire substrate surface, or the cell-containing solution is seeded on the cell affinity region pinpointed by an injector or the like.
The current problem is that the cell exclusion surface using chemical substances is unstable over time, and the cell exclusion ability is weakened and the cell pattern is lost in a short period of time. The present invention is effective not only in suppressing initial adhesion of cells but also in suppressing subsequent growth and migration by providing cell exclusion ability to the geometric structure constructed on the substrate surface. The pattern is maintained, which is effective for improving the accuracy of cell chip analysis, improving the biocompatibility of implants, and enhancing the advancement of stem cell engineering in regenerative medicine.

  EXAMPLES Next, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples.

Example 1
Our research results on the effect of surface roughness on cell adhesion, proliferation, and differentiation of mouse-derived osteoblast-like cells (MC3T3-E1 cells) indicate that cell growth and differentiation can be controlled by the micrometer-order geometry of the substrate surface. Thought. Based on this idea, the purpose of investigating how the shape and dimensions of the geometric structure created on the surface of the substrate affects cell adhesion, proliferation, extension, and migration is to be performed on the surface of the glass slide by the sandblast method. Four types of grooves having a width of 50, 100, 150 and 300 μm and a depth of 100 μm were formed, and an island structure of a cell adhesion region surrounded by the grooves was produced. As shown in FIGS. 2A-1 and B-1, 250 μm square island structures were formed in an array.
When sandblasting is performed with a groove depth of 100 μm and a groove width of 300 μm (aspect ratio = groove depth ÷ groove width = 0.33), the cross section of the groove is U-shaped as shown in FIG. The radius of curvature was 206 μm. Even when the groove depth was 100 μm and the groove width was 150 μm (aspect ratio = 0.67), it was U-shaped and the radius of curvature was 51 μm. On the other hand, when the groove depth was 100 μm and the groove width was 50 μm (aspect ratio = 2), the groove shape was V-shaped with a depression angle = 28 degrees as shown in FIG. 2A-2. The curvature radius of the bottom surface was 8.5 μm. Even when the groove depth was 100 μm and the groove width was 100 μm (aspect ratio = 1), a V-shape with a depression angle = 53 degrees was obtained. In this case, the curvature radius of the bottom surface was 16.2 μm.
In the following cell culture experiment, using two types of substrates with aspect ratio = 2 (V-shaped groove) and aspect ratio = 0.33 (U-shaped groove), the shape of the groove for cell response (cell adhesion, proliferation, differentiation) The effect of was examined.

Example 2
The initial adhesiveness of MC3T3-E1 cells on the substrate on which two types of V-shaped and U-shaped grooves formed in Example 1 were formed was examined.
Cells were seeded on a substrate in a state suspended in a culture medium alpha-Minimum Essential Medium (α-MEM). On the first day of the culture, nuclear staining of cells adhered to the substrate was performed using the nuclear staining reagent DAPI (6-diamidino-2-phenylindole dihydrochloride) to examine the distribution of adherent cells.
FIGS. 3A-1 and B-1 show photomicrograph images focused on the surface of the island structure surrounded by the groove shape. Fine spots in the photographic image are stained cell nuclei. On the island structure (A-1) of the cell adhesion region surrounded by the V-shaped groove and the island structure (B-1) of the cell adhesion region surrounded by the U-shaped groove, the cells appear to be the same. It is shown that it adheres to.
On the other hand, focusing on the groove part and observing the adherent cells in the groove part, according to FIGS. 3A-2 and B-2, in the case of the U-shaped groove structure with an aspect ratio = 0.33 (B-2), quite a lot Although the cells are initially attached to the groove portion, it can be seen that when the aspect ratio = 2 and the V-shaped groove structure is (A-2), almost no cells are initially attached to the groove structure portion.
From these results, cells seeded as a cell suspension hardly adhere to the V-groove, so placing the V-groove around the cell adhesion region forms the desired cell pattern on the substrate. It became clear that it was convenient for. One of the reasons why it is difficult for cells to adhere to the V-shaped groove is because the structure narrows toward the bottom, so it is difficult for the culture medium filling the V-shaped groove and the cell suspension added later to occur. It is possible that the access of cells to the V-shaped groove is hindered.

Example 3
In order to investigate whether the initial adhesion pattern formed on the first day after cell seeding was developed and maintained thereafter, the cells were stained with DAPI on the 14th day of culture, and the cell distribution on the substrate was examined. .
As shown in Fig. 4, the island structure (A-1) of the cytophilic region surrounded by the V-shaped groove and the island structure (B-1) of the cytophilic region surrounded by the U-shaped groove In the cells, and in the cells of the U-shaped groove part (B-2) having an aspect ratio = 0.33, the nuclei were stained, and the cells were found to be normal because they were circular. However, for the V-groove portion (A-2) cells with an aspect ratio = 2, the entire cytoplasm was stained with DAPI, and it was observed that the cells were necrotic with few “healthy” nuclei.
From these results, the cells pushed into the narrow V-shaped groove space are likely to be necrotized due to uneven stress applied to the cell membrane from the V-shaped groove wall, while the cells in the cytophilic region are flat on the plane. By proliferating, it was shown that the initial adhesion pattern developed over the course of the culture and the cell pattern was maintained for at least 14 days.

Example 4
On the 7th day after cell seeding, MC3T3-E1 cells on the substrate undergo differentiation by changing from the growth medium α-MEM to the differentiation medium supplemented with differentiation factors, and alkaline phosphatase (ALP), osteocalcin , The osteoblast differentiation marker protein such as parathyroid hormone receptor is expressed. The degree of differentiation of MC3T3-E1 cells on the 14th day substrate was examined by ALP positive cell staining.
As shown in FIG. 5, on the island structure (A-1) of the cytophilic region surrounded by the V-shaped groove and on the island structure (B-1) of the cytophilic region surrounded by the U-shaped groove. The cells were positive for ALP, indicating that bone differentiation was progressing.
On the other hand, the cells in the V-shaped groove part (A-2) with an aspect ratio = 2 were not stained with hematoxylin and were found to be in a state of cell necrosis. The cells in the U-shaped groove part (B-2) with an aspect ratio = 0.33 were stained with hematoxylin, and it was found that although the degree of differentiation was low, the cell function was maintained.
From these results, it was shown that the cells on the island structure of the cytophilic region surrounded by the V-shaped groove maintained the ability to grow and differentiate normally.

  The cell substrate on which the cell affinity region of the present invention and the cell-exclusion region of the V-shaped groove structure are arranged can stably express cell exclusion for a long period of time by the V-shaped groove structure. Is a promising platform for creating new cell chips and is applicable to basic and applied biology. Furthermore, this cell substrate technology can be applied not only to cell chips, but also to the surface of implant materials and as a culture substrate for the production of cell sheets for regenerative medicine. It can also be applied to manufacture various biomedical devices that come into contact with cells in the body.

Claims (9)

  A V-shaped groove that inhibits cell adhesion, proliferation, extension, and migration, and has a depth and width that allow spherical cells suspended in the culture medium to enter completely. A cell-exclusion groove structure characterized by being less than or equal to the degree.   The cell-excluded groove structure according to claim 1, wherein the curvature radius of the groove bottom is 20 μm or less.   A cell culture substrate on which the cell-exclusion groove structure according to claim 1 or 2 is formed.   The cell culture substrate according to claim 3, wherein a cell-exclusion groove structure is formed around a cell affinity region.   The cell culture substrate according to claim 3 or 4, wherein the cell-exclusion groove structure is provided in a single row or a plurality of rows.   The cell culture substrate according to any one of claims 3 to 5, wherein the cell affinity region is a smooth plane.   The cell culture substrate according to any one of claims 3 to 6, wherein the cell affinity region is surface-modified with a cell adhesive protein.   The cell culture substrate according to any one of claims 3 to 7, wherein the cell culture substrate is used as a cell chip, an implant material, or a cell sheet culture substrate.   3. The method for producing a cell-excluded groove structure according to claim 1, wherein the groove is processed by any one of cutting, focused ion beam, sand blasting, injection molding, contact imprint, and lithography.

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