KR20190050010A - Method for controlling microfilament alignment in structure by pre-strain combination and physical properties - Google Patents

Abstract

The present specification relates to a method of aligning microfilaments and a structure therefor. According to an elastic substrate, device, or method of one aspect of the present invention, microfilaments can be aligned in various directions by stretching or compressing and restoring the elastic substrate, and accordingly, cells can be arranged in various and uniform orientations or patterns. By using one aspect of the present invention, a cell culture model used in various studies can be mass-produced more easily.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for controlling alignment of microfibers in a structure according to the combination of line strain and physical properties of the microfibers.

The present specification relates to a structure for aligning fine fibers and a method for aligning the fine fibers.

All the organs and tissues in the body are produced through the developmental stage in the embryonic state, and the cells and extracellular matrix are arranged in a characteristic pattern. These structural features enable the cells located within each organ and tissue to interact with other cells or extracellular matrix in the periphery, and these interactions play a crucial role in the functioning of each organ. As a representative example, the brain, heart, central and distal nerve bundles, and various muscles show a very characteristic structure, and when such a structure breaks down, it causes severe developmental disorders.

In order to study these cell and tissue characteristics more closely, the technical concept of tissue engineering in which cells are planted and cultured in a living body hydrogel to induce them to grow in a similar manner to a specific tissue, is introduced for the first time in Science journal in 1993 , And then animal cell three-dimensional culture technology, which is a method of culturing cells in various synthetic or natural polymer biomaterials, continues to be developed in advanced countries.

For the organs such as liver, cartilage, and kidney, attempts to create a physiological model in a more similar environment than in vivo (in vivo) are somewhat successful. In addition to the normal long-term model, the three-dimensional culture technique was introduced to construct a pathological model such as simulating the intracellular microenvironment in cancer tissues, and the cell-cell, cell-extracellular matrix In-depth research is also proceeding steadily. In the mid-2000s, the application field expanded further, and three-dimensional culture of stem cells was attempted. In particular, in September 2011, the Defense Advanced Research Projects Agency (DARPA) at the National Institute of Health (NIH) And the Food and Drug Administration (FDA) have announced plans to begin a $ 70 million Tissue Chip Project for five years.

As a result, along with the previously developed cell / tissue chip technologies, the related fields are expected to be activated and developed, but the technology for controlling alignment and orientation of cells and tissues is still insufficient.

PCT Open Publication WO2009 / 073548 A1 (2009.06.11)

The present specification is intended to provide a method of aligning an elastic structure and microfibers that enable alignment of fine fibers, and to provide a method of aligning and culturing cells accordingly.

SUMMARY OF THE INVENTION In order to solve the above problems, the present invention relates to an elastic substrate for microfibre alignment comprising microchannels and one or more voids containing raw materials capable of microfibrillating capability or loading a composition comprising microfibers in one aspect to provide.

In another aspect, the present invention provides an elastic substrate for microfibre alignment comprising a microchannel containing a raw material capable of microfibrillating capability or loading a composition comprising microfibers and two or more substrates having different elastic moduli.

In yet another aspect, the present invention can be a microfiber alignment apparatus comprising an elastic substrate for aligning the microfibers and an apparatus for stretching or compressing the elastic substrate.

According to another aspect of the present invention, there is provided a method for fabricating a microfibre fiber, comprising: (1) holding an elastic substrate for aligning microfibers with a strain in a predetermined direction; (2) loading a microchannel in the elastic substrate with a composition containing microfibers or containing a raw material capable of microfibrillating; And (3) excluding the strain in the constant direction.

In another aspect, the present invention may be a cell culture method using an elastic substrate for microfiber alignment or a microfibre alignment method.

In accordance with one aspect of the present invention, microfibers can be aligned in various directions by stretching or compressing and restoring the elastic substrate, thereby allowing the cells to be aligned in various and constant orientations It is possible to easily mass produce various cell culture models required in various studies by utilizing one aspect of the present invention.

1 illustrates three examples of cavities 201, 202 and microchannels 100, 101 in an elastic substrate according to an aspect of the present invention (the circular or oval in the top view is a cavity, In the center, the rectangle is the cavity, the bold line is the microchannel, the bottom circle is the joint of the two circles, and the middle dumbbell shape is the microchannel).
2 illustrates a plan view and a perspective view of respective elastic substrates 10 according to three examples of cavities 201 and 202 and microchannels 100 and 101 according to an aspect of the present invention.
FIG. 3 shows a top elastic substrate 10 and an apparatus 40 for stretching the top elastic substrate 10 of FIG. 2. The left side shows a state before the elastic substrate is stretched, and the right side shows a state in which the elastic substrate is stretched in the longitudinal direction .
FIG. 4 shows a middle elastic substrate 10 and an apparatus 40 for stretching the middle elastic substrate 10 of FIG. 2. The left side shows a state before the elastic substrate is stretched, and the right side shows a state in which the elastic substrate is stretched in the longitudinal direction will be.
FIG. 5 shows the lower elastic substrate 10 and the device 50 for stretching the lower elastic substrate 10 of FIG. 2. The left side shows a state before stretching the elastic substrate, and the right side shows a state in which the elastic substrate is stretched in the lateral direction .
6 is a cross-sectional view of an elastic substrate according to one aspect of the present invention, (Gray arrow) on the circular cavity portion and a strain direction (red arrow) on the microchannel portion when the strain of the elongation in the longitudinal direction is applied, and the lower side of the drawing shows the method according to one aspect of the present invention (The direction of the solid line arrow indicates the direction of the strain, the solid line arrow indicates the length of the strain, and d indicates the diameter and angle of the cavity) The direction in which the fibers are aligned refers to the angle measured with respect to the longitudinal direction of the microchannel before deformation).
7 is a cross-sectional view of an elastic substrate according to one aspect of the present invention, (Gray arrows) on the polygonal cavity and strain directions (red arrows) on the microchannel portion when the strain of the elongation in the longitudinal direction is applied. The bottom of the drawing shows the method according to one aspect of the present invention (The direction of the solid line arrow indicates the direction of the strain, the length of the solid line arrow indicates the size of the strain, and the angle indicates the direction in which the microfibers are aligned) Refers to the angle measured with respect to the longitudinal direction of the microchannel).
FIG. 8 shows an example of an oggic structure, wherein the stomach is a structure before stretching, and the bottom shows a structure when stretched. In the drawing, a black arrow indicates the direction of elongation strain, Direction and force.
FIG. 9 shows a case where the elastic substrate of FIG. 2 is stretched transversely by using the lower substrate as an example, and the lower end shows a state in which fine fibers of the microchannel 101 are aligned by the method according to one aspect of the present invention (The two independent circles or ovals in the top drawing are the cavity 201 and the dumbbell structure 101 between the cavities is the microchannel).
FIG. 10 shows a case where two elastic substrate portions 20 and 30 having different elastic moduli are joined and compressed. The black square in the left drawing is a microchannel 10, and the thick arrows in the right drawing indicate compression strains Direction.
11 is a view showing a state in which fine fibers are aligned by a method according to one aspect of the present invention in which a compressive strain is applied after joining two elastic substrate portions having different elastic moduli by the method of FIG. 10, (The direction of the red arrow in the upper drawing shows the portion of the microchannel being subjected to the force, and the length of the red arrow indicates the magnitude of the force).
12 is an analysis of the alignment direction by pre-stretching according to one aspect of the present invention by a 3D matrix. In the left drawing, a bold arrow indicates a recovery direction after stretching, and a thick, And the right figure shows the directions of the fine fibers recovered after elongation.
Fig. 13 shows the XY plane in Fig. 12, the left drawing shows the state of linear expansion, the middle drawing shows the recovery state after elongation, and the right figure shows the state of? According to? L / L (The orange solid lines in the left and middle figures indicate the microfibers and their orientation).
FIG. 14 is a diagram showing the YZ plane of FIG. 12, the left drawing shows the state of line extension, the middle drawing shows the state of recovery after extension, and the right side shows the state of φ according to ΔL / L (Green solid lines in the left and middle drawings indicate microfibers and their orientation).
FIG. 15 shows the XZ plane of FIG. 12, the left drawing shows the state of linear expansion, the middle drawing shows the recovery state after extension, and the right side shows the state of φ according to ΔL / L (Blue solid lines in the left and middle drawings indicate the microfibers and their orientation).
16 is an analysis of the alignment direction by pre-compressing according to one aspect of the present invention with a 3D matrix. In FIG. 16, a bold arrow in the left drawing indicates the direction of recovery after compression, And the right figure shows the directions of the fine fibers recovered after elongation.
FIG. 17 shows the XY plane of FIG. 16, the left-hand drawing shows the linear compression state, the middle drawing shows the recovery state after compression, and the right-hand side diagram shows an aspect of? According to? L / L (The orange solid lines in the left and middle figures indicate the microfibers and their orientation).
Fig. 18 is a diagram showing the YZ plane of Fig. 16, the left drawing shows the state of linear compression, the middle drawing shows the recovery state after compression, and the right figure shows the state of phi according to DELTA L / L (Green solid lines in the left and middle drawings indicate microfibers and their orientation).
FIG. 19 shows the XZ plane of FIG. 16, the left-hand drawing shows the linear compression state, the middle drawing shows the recovery state after compression, and the right-hand drawing shows the phase of φ according to ΔL / L (Blue solid lines in the left and middle drawings indicate the microfibers and their orientation).

In the present specification, the term " elasticity " refers to a force or a property which returns to the original state after the structure is deformed by an external force, and corresponds to a term that can be easily recognized by an ordinary technician. More specifically, the width or length of a specific structure (for example, an elastic substrate) is 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 35% or more, 40% At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the original width or length after compression after compression of at least 50%, at least 55%, at least 60%, at least 70% Or more.

As used herein, the term " substrate " is not limited as long as it is a structure made of a material having elasticity. Examples of materials having elasticity include PDMS (polydimethylsiloane), but are not limited thereto, and any material known as a material having elasticity known in the art can be used without limitation.

As used herein, the term " substrate portion " includes a portion that includes the substrate, and includes a structure that constitutes a part of the substrate.

As used herein, " microfibers " refers to fibers having a linear structure in submicrometer units, which may refer to microfine materials that can be elongated, thin, and softly curved. Such microfibers may correspond to synthetic or natural polymer fibers, and may be, for example, biofibers such as collagen fibers or actin fibers.

As used herein, the term " elastic substrate for aligning fine fibers " means a substrate having elasticity as a substrate for aligning fine fibers in a specific direction, and the specific direction may be any direction according to purposes and intentions, Or a plurality of directions, and also includes a direction in which a certain pattern is formed.

In the present specification, " strain " refers to the degree of external force or deformation when an external force acts on an object. That is, the strain includes pressure, compression, tensile force, and the like, and includes a ratio of a length that is elongated or reduced with respect to the original length when an object is stretched or compressed.

As used herein, the term " cavity " means voids, voids, and the like formed in the substrate of the present specification, and the shape is not limited thereto and includes a structure that can be deformed when strain is applied to the substrate. When a strain is applied to the substrate, a strain in a certain direction or in various directions can be adjusted around the cavity.

As used herein, the term " microchannel " refers to a passage having a width or depth of micrometers, and includes a passage containing a raw material capable of forming microfibers or loading a composition containing microfibers. In the present specification, the microchannel is located within the elastic substrate, and there is no limitation in shape or length.

As used herein, the term " Auxetic structures " refers to a structure having such properties as a lattice structure in which an internal structure is appropriately designed, and has a negative pitch and a poisson ratio by expanding when stretched and shrinking upon compression. For typical materials with constant volume before and after deformation, they have a positive prong and a poisson ratio. That is, when stretched in the vertical direction, the volume shrinks in the horizontal direction because the volume is constant. However, in the case of the oggic structure, since it has a negative pitch and a ratio, it expands in the horizontal direction when stretched in the vertical direction, contracts in the horizontal direction when compressed in the vertical direction, Respectively.

As used herein, the term " adhesive " refers to a component or raw material that contains a raw material having fine fiber forming ability or prevents a composition containing fine fibers from adhering to a substrate or a microchannel to move or flow down.

As used herein, the term " gelation " refers to the conversion of a sol into a gel. General characteristics of gelation include a pronounced increase in elasticity of the system and a rapid increase in viscosity.

In particular, the structural characteristics of complex and highly characteristically arranged tissues (eg, brain, nervous tissue, muscle tissue, etc.) are related to the tissue This has a decisive influence on the function. For example, if the neural network is not properly aligned and organized in the embryo, it is known to cause various mental disorders such as autism.

As the problems of cell and tissue culture techniques have been raised up to now, efforts to realize cell and tissue culture in 3-dimensional in vitro environment have begun to become active. The development of technologies that enable the cultivation of cells in an aligned biomaterial structure will continue to increase in demand in the future.

As a representative biomaterial used for cell culture, there is collagen which is the fibrous material which constitutes the largest proportion of extracellular matrix constitution of our body. Conventional techniques for aligning collagen fibers include 1) applying an electric field or 2) applying a magnetic field to an external device, 3) using the property that cells implanted in the collagen contract collagen, or 4) flowing the collagen solution through a narrow tube (Shear force) due to fluid flow are known. Applying electricity or magnetic field through an external device may deteriorate reproducibility and practicality. Cells stimulated by electricity or magnetic field may change physiological activity and cause considerable toxicity. In addition, with the above-mentioned methods, it is difficult to arrange the fibers at the same time while fabricating an integrated three-dimensional structure in various shapes and sizes of different kinds of collagen.

In one aspect, the present invention is an elastic substrate for microfiber alignment capable of meeting the above-mentioned demand and capable of solving the above problems, wherein the substrate comprises a raw material capable of microfibrillating ability or a composition comprising microfibers Microchannel; And one or more voids. ≪ / RTI >

By simply stretching or compressing the substrate and then recovering the substrate, strain can be applied to various portions of the microchannel in various directions, and in consideration of the direction of the strain, alignment directions, patterns, etc. of the microfibers Can be adjusted. Accordingly, the cells to be co-cultured can be cultured and proliferated according to their orientation and pattern.

In one embodiment, the cavity may be arranged in a direction parallel to the microchannel. In one aspect, the cavity may be separate, not connected to the microchannel.

In another embodiment, the cavity may be formed symmetrically or asymmetrically to the left or right or up and down the boundary of the microchannel.

In other embodiments, the cavity may be at least one of circular, elliptical, and polygonal with more than triangles.

In another embodiment, the microchannel may be linear, linear in shape with both ends rounded or elliptical, or linear with both ends polygonal in shape.

In one embodiment, the cavity and the microchannel may be of an Auxetic structure. In one aspect, the euggetic structure may have a negative feed ratio.

In another aspect, the present invention provides an elastic substrate for microfiber alignment, the microchannel comprising a microfibre-forming raw material or loading a composition comprising microfibers; A first substrate portion; And a second substrate portion having a modulus of elasticity different from that of the first substrate portion.

In one embodiment, the two substrate portions may be arranged to be in contact with each other.

In another embodiment, the microchannel may be formed over two substrate portions having different elastic moduli. The substrate portion may be more than two.

In another embodiment, the material of the elastic substrate may be an elastic polymer.

In another embodiment, the elastic polymer is selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins ), Polyimides, polystyrene, cellulose acetate, polyethyleneterephthalate (PETP), natural polyisoprene, synthetic polyisoprene, polybutadiene, chloroprene rubber, butyl rubber, styrene- Butadiene rubber, nitrile rubber, ethylene propylene rubber, ethylene propylene diene rubber, epichlorohydrin rubber, polyacryl rubber, silicone rubber, fluorosilicone rubber, fluoroelastomer, polyether block amide, chlorosulfonated polyethylene, ethylene - in the group consisting of vinyl acetate and polyurethane It may be more than one selected.

In one aspect, the first substrate portion has a weight ratio of the elastic polymer and the curing agent for curing the elastic polymer of 11 or more: 1, 12 or more: 1, 13 or more: 1 or more, 14 or more: 1 or more than 20: 1 or more than 20: 1 or more than 21: 1 or more than 22: 1 or more than 23: 1 or more than 24: 1 or more than 25: 1 or more than 26: Or more: 1 or more than 30: 1 or more than 32: 1 or more than 34: 1 or more than 36: 1 or more than 38: The weight ratio of the elastic polymer and the curing agent for curing the elastic polymer is 40 or less: 1, 38 or less: 1, 36 or less: 1, 34 or less: 1, 32 or less: 1, 30 1 or less: 1 or less: 1 or less: 1 or less: 1 or less: 2 or less: 1 or less: 1 or less: 1 or less: 1 or less than 16: 1 or less than 14: 1 or less than 12: 1 or less than 11: 1.

In one aspect, the second substrate portion has a weight ratio of the elastic polymer and the curing agent for curing the elastic polymer of 5 or more: 1, 6 or more: 1, 7 or more: 1 or 8 or more: 1 or 9 or more: 1 or more than 11: 1 or more than 12: 1 or more than 13: 1 or more than 14: 1 or more than 15: 1 or more than 16: It can be done. The second substrate portion may have a thickness of 20 or less: 1 or less than 19: 1 or less than 18: 1 or less than 17: 1 or less than 16: 1 or less than 15: 1 or less than 14: : 1, 11 or less: 1, 10 or less: 1, 9 or less: 1, 8 or less: 1, 7 or less: 1 or 6 or less:

In one aspect, the elastic modulus (or Young's modulus) of the first substrate portion is 0.1 MPa or more, 0.2 MPa or more, 0.3 MPa or more, 0.4 MPa or more, 0.42 MPa or more, 0.44 MPa or more, 0.46 MPa or more, 0.47 MPa or more, Or more, 0.5 MPa or more, 0.6 MPa or more, 0.7 MPa or more, 0.8 MPa or more, or 0.9 MPa or more. (Or Young's modulus) of the first substrate portion is 2.0 MPa or less, 1 MPa or less, 0.9 MPa or less, 0.8 MPa or less, 0.7 MPa or less, 0.6 MPa or less, 0.5 MPa or less, 0.49 MPa or less, 0.48 MPa or less , 0.47 MPa or less, 0.46 MPa or less, 0.45 MPa or less, 0.44 MPa or less, 0.43 MPa or less, 0.42 MPa or less, 0.4 MPa or less, 0.35 MPa or less, 0.3 MPa or less, 0.25 MPa or less, 0.2 MPa or less or 0.15 MPa or less .

In one aspect, the Young's modulus of the second substrate portion is 1 MPa or more, 1.5 MPa or more, 1.8 MPa or more, 2 MPa or more, 2.2 MPa or more, 2.4 MPa or more, 2.6 MPa or more, 2.8 MPa or more, MPa, 3.4 MPa, 3.6 MPa, 3.8 MPa, 4.0 MPa, 4.5 MPa, 5 MPa, 5.5 MPa, 6 MPa, 6.5 MPa, 7 MPa, 8 MPa or 9 MPa.

(Or Young's modulus) of the second substrate portion is 10 MPa or less, 9 MPa or less, 8 MPa or less, 7 MPa or less, 6 MPa or less, 5 MPa or less, 4 MPa or less, 3 MPa or less, 2 MPa or less, 1.8 MPa or 1.5 MPa or less .

In one embodiment, the ratio of the elastic polymer and the curing agent of the first substrate portion may be different from that of the second substrate portion. In another embodiment, the modulus of elasticity (or Young's modulus) of the first substrate portion may be different from that of the second substrate portion.

In another embodiment, the microchannel may be coated with an adhesive.

In another embodiment, the adhesive may be at least one selected from the group consisting of glutaraldehyde, polyethyleneimine, poly-L-lysine, poly-D-lysine, and polydodamine.

In yet another aspect, the present invention can be a microfiber alignment apparatus comprising an elastic substrate for aligning the microfibers and an apparatus for stretching or compressing the elastic substrate.

According to an aspect of the present invention, there is provided a method for fabricating a microfibre fiber, comprising: (1) holding an elastic substrate for aligning microfibers with a strain in a predetermined direction; (2) loading a microchannel in the elastic substrate with a composition containing microfibers or containing a raw material capable of microfibrillating; And (3) excluding the strain in the constant direction.

In one embodiment, applying a strain in a predetermined direction to the elastic substrate in the step (1) may be to stretch or compress the elastic substrate.

In another embodiment, applying the strain in the predetermined direction to the elastic substrate in the step (1) may be performed at a specific portion of the microchannel in accordance with at least one of the shape, arrangement and elastic modulus of the elastic substrate in the elastic substrate The strain may be applied in a constant direction with respect to the substrate. In one embodiment, a particular portion of the microchannel may be all or a portion of the microchannel.

In one aspect, the constant direction or specific direction may be longitudinal or transverse, and may be a direction parallel or orthogonal to the microchannel before applying strain.

In another embodiment, applying a strain in a certain direction to a specific portion of the microchannel may cause a strain (strain caused by stretching) to be applied to a specific portion of the microchannel in a certain direction It may be to align and propagate the microfibers in a direction perpendicular to the direction of the strain at the specific portion.

In another embodiment, applying a strain in a certain direction to a specific portion of the microchannel may cause a strain (compressive strain, pre-compressed strain) to be applied to a specific portion of the microchannel in a certain direction It may be to align and propagate the microfibers in a direction parallel to the direction of the strain at the specific portion.

In another embodiment, when applying a strain in a certain direction to a specific portion of the microchannel is to apply both stretch by compression and strain by compression to a specific portion of the microchannel, The microfibers may be aligned and propagated in a direction perpendicular to the direction of the strain at the specific portion.

In yet another embodiment, when applying a strain in a certain direction to a specific portion of the microchannel is to apply both strain due to stretching and strain due to compression to a specific portion of the microchannel, It is possible to align and propagate the fine fibers in the direction parallel to the direction of the strain at the specific portion.

In one aspect, the specific portion may refer to the entirety of the microchannel, the whole of the microchannel to which the strain is applied, or a portion of the microchannel to which the strain is applied.

In another embodiment, the strain of step (1) may be applied to the width or length of the elastic substrate so as to be deformed by 5% to 80% based on a value before the strain is applied. If it is less than 5%, the influence by the strain is insignificant. If it exceeds 80%, the elastic substrate may be deformed irrecoverably, and the influence of the strain may be unknown. In one aspect, the strain is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% 60% or more, 65% or more, 70% or more, or 75% or more. In another aspect, the strain is less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35% , 25% or less, 20% or less, 15% or less, or 10% or less.

In another embodiment, the strain in the step (1) may be such that the absolute value of DELTA L / L is 0.001 to 0.8. In this case, for example, L may be the width or length of the elastic substrate before applying the strain, and the DELTA L may be a width or a length as long as the strain is applied. If the absolute value of DELTA L / L is less than 0.001, the effect of strain is insignificant. If the absolute value of DELTA L / L is more than 0.8, there is a possibility that unrecoverable deformation may occur in the elastic substrate, and the influence of strain may be unknown. In one aspect, the absolute value of DELTA L / L may be 0.001 or more, 0.005 or more, 0.01 or more, 0.05 or more, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, or 0.7 or more. In another aspect, the absolute value of DELTA L / L is not more than 0.8, less than 0.75, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1, less than 0.05, less than 0.005, ≪ / RTI >

In another embodiment, the microfibrillating raw material of step (2) is selected from the group consisting of collagen, laminin, fibronectin, entactin, hyaluronic acid, nylon, polyacrylic acid, One selected from the group consisting of polycarbonate, polyurethane, poly (ethylene vinyl acetate), polystyrene, polyvinyl alcohol, cellulose acetate, polyethylene oxide, elastin, gelatin, fibrinogen, fibrin, alginate, cellulose, silk fibroin, chitosan, Or more.

In one embodiment, before the step (1), the step of coating the elastic substrate for fine fiber alignment with an adhesive may be further included.

In another embodiment, the step (2) may further include the step of aggregating the raw material to form a fine fiber nucleate or an aggregate.

In another embodiment, the time for forming the microfiber nucleate or aggregate (e.g., fibril) may be from 5 minutes to 15 minutes. In one aspect, the time may be at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, or at least 14 minutes. In other aspects, the time may be less than 15 minutes, less than 14 minutes, less than 13 minutes, less than 12 minutes, less than 11 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, or less than 6 minutes. As described above, the composition is subjected to a partial gelation process for 5 to 15 minutes (inductive phase, Lag phase) after loading, wherein the microfibre protein is self-assembled to generate short fibrils do. If the induction unit is less than 5 minutes, self-assembly is insufficient and strain is applied before the fibrils are well formed, so that it can not be aligned in a desired pattern or direction. When the induction unit is less than 5 minutes, The fibers are rapidly formed before they are formed, so that they can not be aligned in a desired pattern or direction.

In yet another embodiment, the step of forming the microfine nucleate or aggregate may be a step of partially curing the composition into a gel state.

In another embodiment, step (3) may be a step of completely curing the composition into gel while excluding strain in a specific direction.

In another embodiment, the curing time can be from 15 minutes to 2 hours. In one aspect, the curing time is at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes Or more, 90 minutes or more, 100 minutes or more, or 110 minutes or more. In another aspect, the curing time is less than 2 hours, less than 110 minutes, less than 100 minutes, less than 90 minutes, less than 80 minutes, less than 70 minutes, less than 60 minutes, less than 50 minutes, less than 45 minutes, less than 40 minutes, Less than 25 minutes, or less than 20 minutes. When the curing time is less than 15 minutes, complete curing is not performed and the fine fibers are not maintained in an aligned state. If the curing time is longer than 2 hours, there is a fear that cell growth and growth are not smooth due to excessive curing.

In another aspect, the present invention may be a cell culture method, wherein the composition further comprises at least one of a cell and a stem cell.

In another aspect of the present invention, an elastic substrate comprising microfibers aligned after step (3) is placed in a cell culture medium containing at least one of cell and stem cells, The method further comprising the step of culturing one or more cells.

In one embodiment, the method may be to align and propagate the cells to be cultured in the direction or pattern of alignment of the microfibers.

In another embodiment, the cell may be at least one selected from the group consisting of epithelial cells, nerve cells, glioma cells, muscle cells, fibroblasts, and solid cancer cells.

In another embodiment, the stem cells may be at least one selected from the group consisting of embryonic stem cells, adult stem cells, and induced pluripotent stem cells.

The present invention can be applied to the alignment and culture of all the cells capable of proliferating not only neurons and glia, but also epithelial cells, muscle cells, stem cells, etc., which are very sensitive to environmental changes, and thus are widely used.

(E.g., diameter), shape (circle, square, triangle, pentagon, hexagon, etc.) in the elastic substrate according to one aspect of the present invention, distance between cavities, number of cavities, It is confirmed that the alignment can be controlled also according to, When the microfibers are aligned, propagated, or proliferated in the same manner as the polygons of a circle or a triangle, the microfibrils are affected in various ways depending on their shapes, And that the microfibers are aligned, multiplied, or separated in such a manner that at least one of the size of the cavity (e.g., diameter), the distance between the cavities, the number of cavities, (Fig. 6, Fig. 7, Fig. 9, and Fig. 11). The results are shown in Fig.

In one embodiment, when applying a strain in a certain direction to a specific portion of the microchannel is to apply both stretch by compression and strain by compression to a specific portion of the microchannel, , And aligning and propagating the microfibers in a direction perpendicular to the direction of the larger strain in the specific portion

In another embodiment, when applying a strain in a certain direction to a specific portion of the microchannel is to apply both strain due to stretching and strain due to compression to a specific portion of the microchannel, It is possible to align and propagate the fine fibers in a direction parallel to the direction of a larger strain in the specific portion.

In another embodiment, when applying a strain in a certain direction to a specific portion of the microchannel applies both stretching by strain and compressing strain to a specific portion of the microchannel, the magnitude of the resultant force of the two strains And the alignment and propagation directions of the microfibers in the specific portion can be determined according to the direction and the direction.

In another embodiment, when the resultant is an elongation strain for the particular portion, it may be to align and propagate the microfibers in a direction perpendicular to the direction of the resultant force at the particular portion.

In another embodiment, when the resultant is a compressive strain for the specific portion, the specific portion may be to align and propagate the microfibers in a direction parallel to the direction of the resultant force.

The alignment of the microfibres and cells according to the elastic structure and method was confirmed through actual fluorescent staining, and the alignment direction and the degree of alignment were calculated. The alignment direction and degree of alignment of the fine fibers in the 3D matrix were analyzed and shown in FIG. 12 to FIG. FIGS. 12 to 19 are graphs showing the relationship between the microfibers and the alignment directions of the microfibers and the cells when the gelatinization is completed after the microfluidic channels are loaded with the microfluidic channels, "He said.

12 shows a case where the strain is a strain due to pre-stretching, a thick gray line of the 3D matrix is fine fiber, a black arrow is pre-stretched in the y-axis direction, Is the direction of recovery to the original shape when excluded, l ₀ is the length of the microfibers, and θ is the angle (or ellipse) relative to the x-axis relative to the x axis and φ (lowercase Greek) The angle (or hori- zosity) at which the fine fibers are measured in the z-axis direction. ? is the magnitude of the elastic strain represented by 1 +? _pre , where? _pre is the magnitude of the strain,? L / L, where L is the original length of the elastic substrate measured in the y axis direction of the elastic substrate, ΔL is a positive number if the strain is stretched and ΔL is negative if the strain is compressed). ₀ and _rs represent the strain before and after the strain, respectively, and _yz and _xz represent the projections from the yz plane and the xz plane, respectively (for example, "φ _{0 , yz} " the value of φ when projected from the yz plane). Figs. 13 to 15 are views (Fig. 13, an orange line) projected from the xy plane of the fine fiber, a diagram projected from the yz plane (Fig. 14, green line) 15, and the fine fibers are blue lines). The graphs on the right side of FIGS. 13 to 15 show graphs of how the θ and φ fluctuate with ΔL / L before and after applying strain, respectively, corresponding to the left and middle drawings, and a mathematical expression for calculating θ and φ is derived .

FIG. 16 shows a case where the strain is a strain due to pre-compression. The thick gray line of the 3D matrix is fine fiber, the black arrow is pre-compressed in the y-axis direction, and when sikyeoteul preclude the restoration of the original shape orientation, l ₀ is the length of the fine fibers, on the θ, φ, λ, ε _pre, △ l / l, _{0, rs, yz,} and _xz are 12 to 15 It is like that. 17 to 19 are diagrams showing a state in which fine fibers are projected from the xy plane (Fig. 17, fine lines are orange lines), planes projected from the yz plane (Fig. 18, fine lines are green lines) 19, and the fine fibers are blue lines). 17 to 19, graphs are plotted so that? And? Vary in accordance with? L / L before and after strain is applied to correspond to left and middle drawings, respectively, and mathematical equations for calculating? And? Are derived .

As a result of confirming the sorting direction and degree as described above through actual fluorescent staining, it was found that the alignment direction prediction according to the calculation was successful (see FIGS. 6, 7, 9, and 11).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

[Example 1] Production of elastic substrate

In order to produce an elastic substrate according to one aspect of the present invention, a polydimethylsiloxane (PDMS), a polymethylmethacrylate (PMMA), a polyacrylate, a polycarbonate, a polycylic olefin polycyclic olefins, polyimides, polystyrene, cellulose acetate, polyethyleneterephthalate (PETP), natural polyisoprene, synthetic polyisoprene, polybutadiene, chloroprene rubber, butyl rubber , Styrene-butadiene rubber, nitrile rubber, ethylene propylene rubber, ethylene propylene diene rubber, epichlorohydrin rubber, polyacryl rubber, silicone rubber, fluorosilicone rubber, fluoroelastomer, polyether block amide, Polyethylene, ethylene-vinyl acetate and polyurethane hanes can be used as a raw material. The feature of these raw materials is that when the substrate is made, the hydrogel solution for cell culture can be stretched or compressed in a proper length and in various directions before, during, and after loading The present invention can be carried out with the raw materials because it relates to such elongation, compression or a combination thereof in one aspect. One example of the raw material is PDMS.

PDMS and PDMS curing agent (Sylgard 184, Dow Corning) were mixed at a weight ratio of 10: 1, and after removing the gas, they were put into a master mold. After curing at 80 ° C for 1 hour, the PDMS substrate (PDMS chip) was separated from the mold. The lengths of the width, length, and thickness can be variously formed to include both the following examples and experimental examples.

On the other hand, using a mold capable of patterning an asymmetric circular cavity / symmetrical rectangular cavity with the microchannel, a circular (or elliptical) void (201 in FIGS. 1 and 2), a symmetrical rectangular cavity A PDMS substrate 10 was fabricated by forming a plurality of microchannels 202 and a microchannel of 1 x 20 x 0.4 mm was formed to load the hydrogel solution containing cells (see FIGS. 1 and 2) Top and center drawings, 100, 101). The diameter of the circular (or elliptical) cavity 201 was variously made to be 1 to 10 mm, and the gap and the number of cavities were also varied. The length of one side of the rectangular cavity was varied from 2 to 10 mm.

The voids 201 and the microchannel 101 forming the Auxetic structure are formed on the PDMS substrate 10 in the same manner as the bottom views of FIGS. 1 and 2, So that the hydrogel containing the cells can be loaded into the channel 101.

Thereafter, at least one selected from the group consisting of glutaraldehyde, polyethyleneimine, poly-L-lysine, poly-D-lysine, and polydodamine is added to the PDMS substrate Was used as an adhesive. As an example, PDMS substrates were coated with polydodamine. Specifically, ethanol was mixed with deionized water to prepare a 20% (v / v) ethanol solution. (Tris-HCl, (Sigma, T5941) was prepared with 20% (v / v) ethanol in 100 mM Tris-HCl buffer Buffer solution) was prepared and stored at 4 캜.

In addition, 2 mg / mL of dopamine hydrochloride was prepared. Specifically, 20 mg of dopamine hydrochloride (Sigma, H8505) was added to 9 ml of deionized water and stirred for a sufficient time to allow the dopamine hydrochloride to dissolve. Then, 1 ml of the above 100 mM Tris-HCl buffer was added. After addition, dopamine polymerizes more rapidly with polypodamine. The solution was thoroughly stirred to complete a 2 mg / mL solution of dopamine hydrochloride.

The PDMS substrate was coated with the 2 mg / mL dopamine hydrochloride solution in a dark room for 3 hours. And then washed with deionized water for 20 minutes. The above washing was carried out three times.

The multi-module 3D culture substrate prepared as described above was appropriately trimmed and stored so as to be placed in a cell culture container such as a Petri dish.

[Example 2] Production of hydrogel solution and scaffold

A hydrogel solution (500 μL, 0.25% (w / v), 2.5 mg / ml) was applied to the PDMS substrate.

Specifically, 139 μL (139 mg) of a collagen solution (0.9% (w / v), Corning Company, 354249) was added to a 1 mL microtube. 50 μL of 10 × DMEM (Dulbecco Modified Eagle Medium, Sigma-Aldrich, D2429) and 308 μL of 1 × DMEM (Lonza, 12-604F) were added to the microtube. Slowly stir with a 1ml pipette tip. Then, 3 μL of 0.5 N NaOH was added and slowly poured into the same pipette tip. Up to this point, it was carried out in ice to prevent gelation of collagen. In addition to collagen as a raw material of the hydrogel solution, a fibrous material such as collagen (for example, fibrin) and a fibrous material produced through electrospinning (for example, Polyacrylic acid, polycarbonate, polyurethane, poly (ethylenevinylacetate), polystyrene, polyvinyl alcohol, cellulose, polyvinylpyrrolidone, polyvinylpyrrolidone, laminin, fibronectin, entactin, hyaluronic acid, nylon, It is possible to use at least one selected from the group consisting of acetate, polyethylene oxide, elastin, gelatin, fibrinogen, fibrin, agarose, alginate, cellulose, silk fibroin, chitosan and actin, , The design of the experiment, the type of the target cell, and the like.

The hydrogel solution prepared by the above method or the cell-seeded collagen containing the solution was loaded on a PDMS substrate and then gelated at 37 ° C for 30 minutes to 1 hour. Since it is desirable to maintain a humid environment to prevent dehydration of the hydrogel, a wet tissue is placed around the PDMS substrate in the Petri dish and the lid is closed. For the 3D culture of the cells introduced into the hydrogel, gelation was carried out in a cell culture incubator (37 ° C, carbon dioxide 5%).

The hydrogel scaffolds thus prepared were stored in phosphate buffered saline (PBS) or a cell culture medium.

[Example 3] Dyeing for fluorescence analysis

Labeling was performed for visual confirmation of collagen fibers via confocal fluorescence microscopy (inverted confocal laser scanning microscope, LSM 700, Carl Zeiss, Germany). Specifically, a hydrogel solution (for example, 2 mg / ml of collagen) in 0.1 M sodium bicarbonate buffer (pH 9) was dissolved in dimethyl sulfoxide (DMSO) using tetramethylrhodamine (TRITC; Life Technologies) And mixed with 10 mg / ml TRITC. The mixture was then stirred in a dark room at 4 [deg.] C for 24 hours. Unbound TRITC molecules were removed by dialysis with 0.1% [w / v] acetic acid at 4 ° C. The hydrogel solution labeled in this way was applied to Example 2 above.

The labeling may be performed using 5- (and-6) -Carboxytetramethylrhodamine succinimidyl ester. Specifically, a solution of 5- (and-6) -Carboxytetramethylrhodamine succinimidyl ester (5 (6) -TAMRA, SE, mixed isomers; TAMRA, SE; Invitrogen, C1171) was mixed with DMSO to prepare 100 mM TAMRA. Lt; / RTI > And diluted with PBS to make 50 [mu] M TAMRA. The 50 μM TAMRA solution was applied to the gelled hydrogel scaffold in Example 2 and stained in the dark for 1 hour. It was then washed three times with PBS (20 min per cycle).

[Example 4] Confirmation of alignment by pre-stretching

A device for stretching the PDMS substrate (40, 50 in Figures 3 to 5. Any device that can apply a constant force to stretch the PDMS substrate, such as a clamping bar, may be used) The growth and restoration were performed to confirm the orientation of the hydrogen fibrils.

Specifically, the PDMS substrate 10 (having a thickness of 1 to 10 mm) is stretched (i.e., stretched from the Y-axis length L to L + DELTA L of the substrate) to a desired length (DELTA L, DELTA L> pre-stretching), the hydrogel solution or the solution was loaded with cell-seeded collagen into the microchannel. Partial gelation for 5 to 15 minutes at room temperature (the acidic collagen material consists of a single protein and a short fibril fragment and neutralized by 0.5 N NaOH, by self-assembly of the collagen protein during this time After the PDMS substrate containing the collagen in the sol state is completely gelated (30 minutes to 30 minutes) under 5% carbon dioxide at 37 < 0 > C, 1 hour).

An anisotropic / heterogeneous (compact) / multi-module 3D culture substrate prepared as above was placed in a Petri dish. As a result of confirming the image of the gel fiber using a confocal microscope, it was confirmed that the collagen fiber was uniformly aligned in a specific direction as ΔL / L was larger. In particular, according to the analysis results, It was confirmed that the collagen fibers were aligned in the direction perpendicular to the stretching direction of the PDMS substrate 10 by confirming that the collagen fibers were aligned in the 90 ° and 270 ° directions.

[Example 5] Confirmation of alignment by pre-compressing

The apparatus for compressing the PDMS substrate 10 (40, 50 in Figs. 3 to 5, compressing the PDMS substrate using any mechanism that can apply a constant force to compress the PDMS substrate) And the direction of orientation of the hydrogen fibrils was confirmed by restoration.

Specifically, after compressing the PDMS substrate 10 (thickness of 1 to 10 mm) to a desired length (DELTA L, DELTA L <0) (i.e., compressing the substrate from the length L of the Y axis to L + DELTA L) -compression), the hydrogel solution or the solution containing fibroblasts was loaded onto the microchannel. Partial gelation at room temperature for 5 to 15 minutes (the acidic collagen material is composed of a single protein and short fibril fragment and neutralized by 0.5 N NaOH, self-assembly of the collagen protein during this time and short The PDMS substrate containing the collagen in a sol state was completely gelated (30 minutes to 1 hour) at 37 ° C under 5% carbon dioxide.

An anisotropic / heterogeneous (compact) / multi-module 3D culture substrate prepared as above was placed in a Petri dish. Then, the image of the gel fibers was observed using a confocal fluorescence microscope (3D reconstruction was performed using ZEN 2012 software (Carl Zeiss)). As the substrate was compressed more and the absolute value of ΔL / L became larger, It was confirmed that the collagen fibers were aligned in the 0 ° and 360 ° directions with respect to the compression direction of the PDMS substrate. As a result, it was confirmed that the collagen fibers were aligned in the compression direction of the PDMS substrate And that they are aligned in parallel with each other.

[Experimental Example 1] Elastic structure experiment including circular (or oval) cavity and microchannel

A PDMS substrate 10 including an asymmetric circular (or elliptical) void 201 pattern and a microchannel (1 × 20 × 0.4 mm, 100) was manufactured as shown in the upper diagram of FIG. The specific manufacturing method is as described above, and circular (or elliptical) cavities are prepared by variously changing from 3 to 7 mm (FIG. 6). The spacing and number of cavities were varied as shown in Fig.

Thereafter, the same procedure as in Example 4 was carried out, and pre-stretched by setting? L / L to 0.3. The shape of the microchannel in the linear expansion state is deformed as shown in Fig. 6, and the shape of the microchannel is also deformed in this state. The direction of the strain in each part of the microchannel is schematically illustrated by the red arrow in Fig.

That is, when the direction of alignment of the collagen fibers is 90 degrees or 270 degrees with respect to the direction of the strain (force) received at the portion, when the length direction of the microchannel before stretching is 0 degree, The alignment direction of the fibers was found to be 45 degrees. It was also found that the alignment direction of the collagen fibers in the B region of the microchannels was -45 degrees.

By applying this, it is possible to adjust the direction and size of the force applied to each part of the microchannel according to the diameter, spacing, and number of the circular (or elliptical) cavities, and accordingly, the alignment of the collagen fibers can be adjusted in a desired direction, When cells are cultured, they can control the direction of alignment and proliferation.

[Experimental Example 2] Elastic structure test including polygonal cavity and microchannel

A PDMS substrate 10 including a symmetrical rectangular void pattern 202 and microchannels (1 × 20 × 0.4 mm, 100) was manufactured as shown in the middle diagram of FIG. The specific manufacturing method is the same as described above, and the square cavity is formed in a square shape with various lengths of one side of 3 to 7 mm.

Thereafter, the same procedure as in Example 4 was carried out, and ΔL / L was pre-stretched at 0.3 to 0.5 (FIG. 4). The shape of the microchannel in the linear expansion state is deformed as shown in FIG. 7, and the direction and magnitude of the strain can be schematically expressed by the direction and the size of the arrow in this state. It can be seen that the direction and size of the strain (force) with respect to each region of the microchannels are changed when the preheated length becomes long, that is, when the pulling force becomes large (see a red arrow in FIG. 7).

Since the alignment direction of the collagen fibers in a specific position (area) is 90 degrees or 270 degrees in the line extension direction at the specific position (area), when the DELTA L / L is 0.3, , It was found that the alignment direction of the collagen fibers in the C region of FIG. 7 was 90 degrees or 270 degrees with respect to the elongation direction, and 0 degrees with respect to the longitudinal direction of the microchannel. That is, in the region C in FIG. 7, the direction of the force of the linear elongation dominantly influenced. Also, in FIG. 7, it is found that the D region of the microchannel has almost no influence due to the linear elongation and is randomly random.

On the other hand, when? L / L is increased to 0.5, when the longitudinal direction of the microchannel is 0 degree, the alignment direction of the collagen fibers in the C1 region is 90 degrees or 270 degrees with respect to the elongation direction, And it was found that the direction of the force of the linear elongation was dominantly influenced. It was also found that the alignment direction of the collagen fibers in the C2 region was 70 degrees with respect to the longitudinal direction of the microchannel and aligned in the direction perpendicular to the red arrow direction of the force (resultant force), and the collagen fibers in the C3 region It can be seen that the alignment direction is -70 degrees with respect to the longitudinal direction of the microchannel and aligned in the direction perpendicular to the red arrow direction of the force (resultant force). When ΔL / L is increased to 0.5, in the case of the D region, a pre-compressed effect occurs rather than a linear extension as shown in the right-hand side of FIG. 7, 90 degrees to the longitudinal direction of the channel).

By applying this, it is possible to control the direction of the force acting on the microchannel according to the size of the cavity, the magnitude of the force, and the positional relationship between the microchannel and the cavity, and thereby the alignment of the collagen fiber can be adjusted to a desired direction, It is possible to control the direction of alignment and proliferation.

[Experimental Example 3] Elasto-structure test of an oggetic structure

For typical materials with constant volume before and after deformation, they have a positive prong and a poisson ratio. When stretched in the horizontal direction, the volume shrinks in the vertical direction because the volume is constant. However, in the case of the lattice structure in which the internal structure is appropriately designed, the structure is expanded in the vertical direction when stretched in the horizontal direction and contracted in the vertical direction when compressed in the horizontal direction. (Auxetic structure). An example of the structure having the structure shown in the bottom view of Fig. 8, Fig. 1 and Fig. 2 may be mentioned.

In the elastic substrate 10 according to one aspect of the present invention, the cavity of the oak-like structure is formed to confirm the fiber alignment direction. Specifically, a PDMS substrate 10 including a void 201 and a microchannel 101 was manufactured as shown in the bottom view of FIG. The upper and lower cavities (the white circles of the black rim) are made of microcrystalline silicon, which is made of microcrystalline silicon, (Dumbbell shape portion) as a reference and diameters varying from 3 to 7 mm. The microchannel included a channel itself (1 x 20 x 0.4 mm) and a circular (or elliptical) well at both ends (diameter equal to cavity and depth equal to channel section).

Thereafter, the same procedure as in Example 4 was carried out, and pre-stretched by setting? L / L to 0.3. The shape of the microchannel in the linear expansion state is deformed as shown in the upper part of FIG. 9, and the direction and magnitude of the strain can be schematically represented by the direction and the size of the arrow in this state. That is, it was confirmed that the circular (or elliptical) well region at both ends of the microchannel was aligned at 90 degrees with respect to the line stretching direction, and the middle region instead of the circular (or elliptical) (That is, aligned at zero degrees with respect to the microchannel length direction) because they extend in the longitudinal direction without contraction in the longitudinal direction.

By applying this structure, it is possible to control the direction of the force acting on the microchannels by arranging the cavities in the oggetic structure, thereby adjusting the alignment of the collagen fibers in a desired direction, It is possible to control the directionality.

[Experimental Example 4] Experiments of elastic structures having different elastic moduli

We changed the physical properties of the elastic substrate and confirmed the alignment direction of the fibers. If the chip is fabricated with two compositions with different elastic modulus due to the different mixing ratios of the raw material and the hardener, the strain applied to each part (soft, stiff) may be different even if the same strain is applied to the PDMS substrate. That is, the effect of the modulus of elasticity on the fiber alignment was determined by changing the kind of the raw material of the elastic substrate or changing the weight ratio of the raw material to the curing agent.

Specifically, two elastic substrate portions (first substrate portion 20 and second substrate portion 30) having different elastic modulus (particularly, Young's modulus) are bonded as shown in FIG. 10, (10). The ratio of the raw substrate to the curing agent of the elastic substrate was set to 10: 1 (stiff substrate, Young's modulus: 3 MPa), and the other elastic substrate portion had the ratio of the raw substrate to the curing agent ) (Soft substrate, Young's modulus is 0.45 MPa). In addition, the elastic modulus may be changed by changing the raw material of the elastic substrate itself or by changing the curing conditions, instead of the ratio of the raw material to the curing agent. For example, the soft substrate may be a substrate made of PDMS as a raw material, and the stiff substrate may be an ultraviolet hardening polymer (for example, SU-8, polyurethane acrylate (PUA), polyester acrylate, But it is not limited thereto, and either a radical polymerization type or a cationic polymerization type can be used).

Then, the two bonded substrates were regarded as one substrate, and the linear stretching or the linear compression test was carried out as in the case of Example 4 and Example 5. In the case of the linear compressing, the horizontal direction was performed, and the absolute value of DELTA L / Was set to 0.3. As a result, it was confirmed that the effect of the linear pressure on the microchannel part in the case of the soft substrate was aligned to 0 parallel to the compression direction, that is, the longitudinal direction of the microchannel. On the other hand, it was confirmed that the stiff substrate was hardly affected by line compression. However, it can be seen that when the absolute value of DELTA L / L is varied, the effect on the stiff substrate can be affected to some degree, and the soft portion will be more influenced, and thus the direction or tendency of alignment can be controlled (Fig. 11).

The linear extension was performed in the transverse direction, and the absolute value of ΔL / L was set to 0.3. As a result, it was confirmed that the effect of the linear stretch on the microchannel part of the soft substrate was aligned 90 degrees with respect to the direction orthogonal to the stretching direction, that is, the microchannel length direction. On the other hand, it was confirmed that the stiff substrate was hardly affected by the line extension. However, it can be seen that when ΔL / L is different, the effect on the stiff substrate can be affected to some degree, and the soft part will be more influenced, and accordingly, the alignment direction or the tendency of alignment can be controlled.

The above Examples and Experiments were carried out on collagen and fibroblasts (for example, fibroblast 3T3 cell line), but it is also possible to use fibers having a linear structure in a fine unit such as collagen, which are long, thin and softly bendable Even if the natural polymer fibers are used, the result is that the fine fibers are aligned. If the cells can be cultured in vitro or in addition to the fibroblasts, the same result can be obtained that the cells are aligned together It will be possible.

10: substrate
20: first substrate portion
30: second substrate portion
40, 50: Extension device or compression device
100, 101: Microchannel
201, 202: joint

Claims (27)

An elastic substrate for fine fiber alignment,
A microchannel containing a raw material capable of microfibril forming ability or loading a composition comprising microfibers; And
An elastic substrate for microfiber alignment, comprising at least one void.
The elastic substrate of claim 1, wherein the cavity is separate from the microchannels and is arranged in a direction parallel to the microchannels.
The elastic substrate for microfiber alignment according to claim 1, wherein the cavity is formed symmetrically or asymmetrically to the left or right or top and bottom of the microchannel.
The elastic substrate of claim 1, wherein the cavity is at least one of a circular, elliptical, and polygonal triangle or greater.
2. The elastic substrate according to claim 1, wherein the microchannel is linear, the shape of both ends being circular or elliptical, or the shape of both ends being a polygonal shape of triangular or more.
6. The elastic substrate of claim 5, wherein the cavity and the microchannel form an Auxetic structure.
An elastic substrate for fine fiber alignment,
A microchannel containing a raw material capable of microfibril forming ability or loading a composition comprising microfibers;
A first substrate portion; And
And a second substrate portion having a different elastic modulus from the first substrate portion.
8. The elastic substrate according to claim 7, wherein the first substrate portion and the second substrate portion are arranged to be in contact with each other, and the microchannel is formed across the two substrate portions.
The elastic substrate according to any one of claims 1 to 8, wherein the material of the two substrate portions is an elastic polymer.
The method of claim 9, wherein the elastic polymer is selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins ), Polyimides, polystyrene, cellulose acetate, polyethyleneterephthalate (PETP), natural polyisoprene, synthetic polyisoprene, polybutadiene, chloroprene rubber, butyl rubber, styrene- Butadiene rubber, nitrile rubber, ethylene propylene rubber, ethylene propylene diene rubber, epichlorohydrin rubber, polyacryl rubber, silicone rubber, fluorosilicone rubber, fluoroelastomer, polyether block amide, chlorosulfonated polyethylene, ethylene - selected from the group consisting of vinyl acetate and polyurethane At least one elastic substrate for microfiber alignment.
9. The elastic substrate according to any one of claims 1 to 8, wherein the microchannel is coated with an adhesive.
12. The elastic substrate according to claim 11, wherein the adhesive is at least one selected from the group consisting of glutaraldehyde, polyethyleneimine, poly-L-lysine, poly-D-lysine and polydodamine.
9. An apparatus for aligning fine fibers, comprising an elastic substrate for aligning microfibers according to any one of claims 1 to 8 and an apparatus for stretching or compressing said elastic substrate.
(1) holding the elastic substrate for aligning microfibers according to any one of claims 1 to 8 in a state in which strain is applied in a predetermined direction;
(2) loading a microchannel in the elastic substrate with a composition containing microfibers or containing a raw material capable of microfibrillating; And
(3) excluding the strain
&Lt; / RTI &gt;
15. The method according to claim 14, wherein applying a strain in a predetermined direction to the elastic substrate in step (1) stretches or compresses the elastic substrate by a predetermined length.
15. The method of claim 14, wherein applying the strain in the predetermined direction to the elastic substrate in the step (1) is performed in accordance with at least one of the shape and arrangement of cavities in the elastic substrate and the elastic modulus of the elastic substrate. And applying a strain in a certain direction to the portion.
The method according to claim 16, wherein, when applying a strain in a certain direction to all or a part of the microchannel is to apply a strain caused by stretching to all or a part of the microchannel in a predetermined direction, Wherein the microfibers are aligned and propagated in a direction perpendicular to the direction of the strain.
17. The method according to claim 16, wherein, when applying a strain in a certain direction to all or a part of the microchannel is to apply a strain due to compressing to all or a part of the microchannel in a predetermined direction, Wherein the microfibers are aligned and propagated in a direction parallel to the direction of the strain.
15. The method of claim 14, wherein the strain of step (1) is applied to the width or length of the elastic substrate such that it is deformed by 5% to 80% based on the amount before the strain is applied.
15. The method of claim 14, wherein the strain of step (1) is applied so that the absolute value of DELTA L / L is 0.001 to 0.8.
(L is the width or length of the elastic substrate before applying the strain, and DELTA L is the width or length as changed after applying the strain)
The method according to claim 14, wherein the fine fiber forming raw material in step (2) is selected from the group consisting of collagen, laminin, fibronectin, entactin, hyaluronic acid, nylon, polyacrylic acid Selected from the group consisting of polyvinyl chloride, polycarbonate, polyurethane, poly (ethylene vinyl acetate), polystyrene, polyvinyl alcohol, cellulose acetate, polyethylene oxide, elastin, gelatin, fibrinogen, fibrin, alginate, cellulose, silk fibroin, chitosan, At least one microfiber array.
15. The method of claim 14, further comprising, after step (2), partially gelating the composition and integrating the raw material to form a microfiber nucleate or aggregate. Way.
The fine fiber sorting method according to claim 22, wherein the forming time is 5 minutes to 15 minutes in the step of forming the fine fiber nucleate or aggregate.
15. The method of claim 14, wherein step (3) is a step of completely curing the composition to gel while excluding strain in a certain direction.
25. The method of claim 24, wherein the time required for curing is from 15 minutes to 2 hours.
14. The cell culture method according to claim 14, wherein said composition further comprises at least one of a cell and a stem cell.
An elastic substrate comprising microfibers arranged after step (3) of claim 14 is placed in a cell culture medium containing at least one of cells and stem cells to culture at least one of the cells and stem cells &Lt; / RTI &gt;

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