WO2014126228A1 - Hydrogel substrate for cell evaluation, and cell evaluation method - Google Patents

Abstract

In order to more accurately evaluate the infiltration behavior of cancer cells under conditions approaching the environment in a living body than the prior art, a novel cell culture system, which is a three-dimensional environment and in which a plurality of types of cells co-exist, is constructed. For that purpose, a hydrogel substrate for cell evaluation, which is constituted by hydrogel and has a fiber- or sheet-like form, is used. The hydrogel substrate has a region (A) inside a cross section (S) in a plane perpendicular to a longitudinal axis, the periphery of which does not touch the periphery of the cross section (S) and which contains first cells. In addition, the region (A) is constituted by hydrogel with a low modulus of elasticity compared to a region (B) outside the region (A) in the cross section (S).

Description

Hydrogel substrate for cell evaluation and cell evaluation method

The present invention relates to a hydrogel substrate for cell evaluation and a cell evaluation method.

In order to develop anti-cancer drugs that are highly effective and selective against cancer and have few side effects, screening operations are necessary to narrow down useful drugs from a large number of new drug candidates. A drug assay using cultured cancer cells has been performed as a pre-stage for evaluating drug efficacy using animals. *

When evaluating the extent to which an anticancer drug acts on cancer cells, it is important to quantitatively evaluate the invasion and metastasis of cancer cells. *

Currently, in order to elucidate the invasion behavior and metastasis of cancer cells, in addition to normal planar culture using a multiwell plate, etc., a culture method using a multiwell plate incorporating a membrane with a hole with a diameter of several microns is incorporated. Measurement of the half-inhibitory concentration (IC50) of an anticancer agent with respect to the survival rate or infiltration degree of cells has been carried out. *

In addition, research on cell culture systems for reproducing the behavior of cancer cells in vivo more faithfully outside the body as compared with single culture on a plane has been actively conducted. As an example, as shown in Patent Document 1, a system for embedding cancer cells at a certain position inside a three-dimensional hydrogel and evaluating the behavior of cancer cells in the gel has been proposed. Infiltration and medicinal efficacy tests in a typical environment are possible. *

In addition, as shown in Non-Patent Document 1, a system for co-culturing cancer cells and other cells has been proposed, although it is a culture on a flat surface. In this system, human cervical cancer cells and human vascular endothelial cells are seeded in a chamber having a valve in the center, thereby co-culturing in a two-dimensional environment, and there are multiple types of cells. The behavior of cancer cells in an environment close to a living tissue can be observed. *

Furthermore, as shown in Non-Patent Document 2, focusing on the fact that the hypoxic region inside the tumor tissue expresses various genes related to the malignancy of the tumor, spherical aggregates of cancer cells (spheroids) A method for reproducing a three-dimensional hypoxic region has been proposed. By seeding and culturing various types of cancer cells on a culture plate that has a lattice-shaped uneven structure on the bottom, a spherical cell aggregate (spheroid) of uniform size is constructed, and the inside of the tumor in the living body The hypoxic region often seen in is reproduced.

JP 2010-110272 A

However, in a normal drug assay using a multiwell plate, cancer cells seeded on a flat culture substrate are used, so that a three-dimensional and in vivo tissue in which multiple types of cells are present at high density It is difficult to say that the environment of the cancer can be reproduced, and it is impossible to accurately evaluate the effect of the anticancer agent under the same conditions as the physiological environment. Therefore, it is necessary to develop a system and method for performing cell evaluation in a cell culture environment closer to a living body. *

In addition, when the technique shown in Patent Document 1 or Non-Patent Document 2 is used, an environment that is partially similar to the biological environment is reproduced in the sense that cancer cell invasion is evaluated in a three-dimensional space. However, there is a problem that it is impossible to accurately evaluate the efficacy of an anticancer drug because it is very different from a tumor in a living body in which cancer cells and normal cells interact with each other. *

Further, when the technique shown in Non-Patent Document 1 is used, although it is similar to the environment in the living body in the sense that there are a plurality of types of cells, it is a two-dimensional planar cell culture system. There is a problem that a correlation with a three-dimensional tumor tissue cannot be obtained. *

In other words, in order to evaluate the behavior of cancer cells under conditions that are closer to the in vivo environment, (1) in the three-dimensional environment, (2) multiple types of cells can be shared. Although it is desirable to construct a system for culturing, a system that satisfies such conditions has not been developed so far. *

Furthermore, in evaluating the degree of cancer cell invasion, if the initial position of the cancer cell can be controlled to a certain level, it is very advantageous to quantitatively and accurately assess the invasion of cancer cells. So far, a method for arranging cells inside a three-dimensional culture environment has hardly been developed. *

The present invention has been made in view of the above-mentioned problems of the prior art, and its object is to accurately detect cancer under conditions closer to a physiological environment than the prior art. In order to evaluate the infiltration behavior of cells, a new cell culture system that is a three-dimensional environment and coexists with a plurality of types of cells is to be constructed. *

The present invention also provides a novel cell culture system that enables quantitative evaluation of cancer cell invasion in a very simple and accurate manner by limiting the initial position of cancer cells to a certain range. Is. *

Furthermore, the present invention is to provide a novel cell evaluation method that enables cancer cell invasion evaluation in an environment similar to that in vivo by using the prepared novel cell culture substrate in a simple and accurate manner. It is what.

In order to achieve the above object, an invention according to one aspect of the present invention includes a hydrogel, a fiber-like or sheet-like form, and a cross section in a plane perpendicular to the longitudinal axis. Inside S, the peripheral edge is not in contact with the peripheral edge of the cross section S, and the first cell is present, and the area A is an area B other than the area A in the cross section S. It is a hydrogel base material for cell evaluation comprised by the hydrogel with a low elasticity modulus compared with. By using such a base material, it is possible to place the first cell to be evaluated, such as a cancer cell, at a specific position inside the three-dimensional fiber-like or sheet-like hydrogel base material. In addition, the region and direction in which the first cells infiltrate and proliferate can be limited to only a certain region inside, and an accurate infiltration behavior can be evaluated. *

Further, in the invention according to this aspect, although not limited, the hydrogel base material partially touches the region A and the region B in the cross section S and partially on the peripheral edge of the cross section S. The region C is in contact with the region C, and the region C is preferably made of a hydrogel having a lower elastic modulus than the region B. By doing in this way, it is possible to control the direction in which the first cell to be evaluated infiltrated in the hydrogel base material is infiltrated from the region A to the outer side of the hydrogel base material through the region C. Thus, it becomes possible to evaluate the infiltration behavior of the first cell embedded in the region A more accurately and simply. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is desirable for the area | region B to have a 2nd cell different from said 1st cell. By doing so, for example, in the environment where cells other than cancer cells exist around the region A where the cancer cells exist as the first cells, and the cancer cells interact with the cancer cells, Since the situation of infiltration toward the outside of the material can be reproduced, the infiltration behavior of cancer cells can be accurately and easily evaluated. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is desirable for the area | region C to have said 2nd cell. By doing so, it is possible to reproduce the situation where cells other than cancer cells exist in the region that is the invasion direction of cancer cells, so that the invasion behavior of cancer cells can be accurately and easily evaluated. Become. *

Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable that the hydrogel which comprises the area | region A, the area | region B, and the area | region C is comprised with the derivative of alginic acid or alginic acid. By doing so, it is easy to mold, has excellent biocompatibility, can embed cells while maintaining cell survival, has high mechanical strength, and is stable for a relatively long time in a cell culture solution. A hydrogel substrate can be used. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable that the hydrogel which comprises the area | region A and the area | region C contains propylene glycol alginate. By doing so, it becomes possible to configure the region A and the region C having a low elastic modulus, and the first cells pass from the region A through the region C and out of the hydrogel substrate more efficiently. It becomes possible to infiltrate. *

In the invention according to this aspect, the hydrogel matrix that constitutes the regions A to C is not limited to the proteins constituting the extracellular matrix or the cell adhesion peptide molecules. It is preferable that at least one of them is included. In this way, it becomes possible to evaluate the infiltration behavior of cells in an environment that mimics the biological environment containing a matrix component such as collagen, and the adhesion of cells to the matrix is improved. Infiltration is promoted, and the time required for evaluation can be greatly shortened. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable that said 1st cell is a cancer cell. By doing in this way, provision of the hydrogel base material for evaluating the invasion of a cancer cell is attained. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable that said 2nd cell is cells other than a cancer cell. By doing so, it is possible to evaluate the invasion behavior of cancer cells in an environment similar to the biological environment in which cancer cells and normal cells exist and interact with each other. *

Further, in the invention according to this aspect, although not limited, the second cell is fibroblast, endothelial cell, epithelial cell, smooth muscle cell, skeletal muscle cell, mesothelial cell, parenchymal cell, It is preferably at least one of glandular cells, epidermal cells, nerve cells, osteoblasts, precursor cells of these cells, and cells differentiated from various stem cells. By doing so, it is possible to evaluate the invasion behavior of cancer cells in an environment similar to the biological environment in which these cells interact with cancer cells. *

Further, in the invention according to the present aspect, but are not limited to, the area of the region A, the average diameter of said first cells when is D, it is preferable that the 10D ² or less. By doing in this way, since it becomes possible to restrict | limit the initial position of the 1st cell embedded to the area | region A to a fixed range, its infiltration behavior can be evaluated more correctly and easily. Become.

In the invention according to this aspect, although not limited, if the area of the region A is S and the peripheral length of the region A is L, the circularity 4πS / L ² of the region A is 0.5 or more. Preferably there is. In this way, the initial position of the first cell embedded in the region A can be limited to a certain range in the cross section, so that the infiltration behavior can be more accurately and easily evaluated. It becomes possible.

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, when the average diameter of said 1st cell is set to D, it is preferable that the width | variety of the area | region C is 3D or less. By doing in this way, since the width | variety of the area | region C which passes when a cancer cell infiltrates outside can be restrict | limited, the infiltration evaluation of a cancer cell more efficiently and rapidly becomes possible. *

Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable that a hydrogel base material is a fiber form 500 micrometers or less in diameter. In this way, it becomes possible to prevent necrosis of cells embedded inside due to lack of oxygen or nutrients, and to reduce the time required for cancer cells to infiltrate outside the hydrogel substrate. It can be shortened.

Further, in the invention according to this aspect, although not limited, the hydrogel base material is a sheet having a thickness of 500 micrometers or less, and a plurality of regions A exist in the cross section S. Also good. By doing so, as in the case of the fiber-like hydrogel substrate, it becomes possible to prevent cell necrosis and shorten the invasion time of cancer cells, and also the region A where the first cells are present. By providing a plurality of cells, it is possible to construct a higher-density cell evaluation system in which cells to be evaluated are arranged in parallel. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable that the density of said 1st cell contained in the area | region A is 100,000 or more per 1 cubic centimeter. By doing so, it becomes possible to use a hydrogel substrate in which cancer cells are embedded at a high density inside, and it is possible to evaluate the invasion behavior of cancer cells more efficiently. *

In the invention according to this aspect, although not limited, the density of the second cells included in the regions B to C is preferably 1 million or more per cubic centimeter. By doing so, it is possible to use a hydrogel base material in which normal cells are embedded at a high density, and it is possible to evaluate the invasion behavior of cancer cells in a situation closer to a biological environment. *

In the invention according to this aspect, the hydrogel substrate is not limited, but the hydrogel base material has at least two inlets I1 to In (n ≧ 2) and inlet channels CI1 to CI1 connected to the inlets I1 to In, respectively. CIn, at least one junction P1 to Pm (m ≧ 1) where the inlet channels CI1 to CIn merge simultaneously or stepwise, a merge channel G existing downstream from the merge points P1 to Pm, and a merge A first aqueous solution containing sodium alginate and in which the first cells are suspended with respect to the flow path structure X having an outlet O existing downstream of the path G is also passed through the inlet I1. A second aqueous solution containing sodium alginate is continuously introduced through the inlet I2, and the first aqueous solution and the second aqueous solution are brought into contact with each other inside the flow channel structure X. X Or by externally bringing the first aqueous solution and the second aqueous solution into contact with the gelling agent aqueous solution, and continuously gelling in a state where the first aqueous solution and the second aqueous solution are in contact with each other. Region A is formed by gelation of the first aqueous solution, and region B is formed by gelation of the second aqueous solution. It is desirable that By doing so, it has a cross-sectional pattern composed of multiple parts with different compositions, the position of the embedded cells is accurately controlled, and the cells can be embedded alive, in the form of fibers or sheets The hydrogel substrate can be easily and continuously produced. *

In the invention according to this aspect, the hydrogel base material is not limited, but the hydrogel base material has at least three inlets I1 to In (n ≧ 3) and inlet channels CI1 to CI1 connected to the inlets I1 to In, respectively. CIn, at least one junction P1 to Pm (m ≧ 1) where the inlet channels CI1 to CIn merge simultaneously or stepwise, a merge channel G existing downstream from the merge points P1 to Pm, and a merge A first aqueous solution containing sodium alginate and in which the first cells are suspended with respect to the flow path structure X having an outlet O existing downstream of the path G is also passed through the inlet I1. A second aqueous solution containing sodium alginate was continuously introduced through the inlet I2 and a third aqueous solution containing sodium alginate through the inlet I3, respectively. The first aqueous solution, the second aqueous solution, and the third aqueous solution are brought into contact with each other, and the first aqueous solution, the second aqueous solution, and the third aqueous solution are disposed inside or outside the flow channel structure X. By making it contact with gelling agent aqueous solution, it produced by continuously gelatinizing in the state which said 1st aqueous solution and said 2nd aqueous solution contacted, Moreover, area | region A is said 1st The aqueous solution is formed by gelation, the region B is formed by the gelation of the second aqueous solution, and the region C is gelled by the third aqueous solution. It may be formed by doing. By doing in this way, it becomes possible to utilize the hydrogel base material by which a cross section is comprised by the at least 3 area | region from which a composition differs. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable that said 2nd cell is suspended in said 2nd aqueous solution thru | or said 3rd aqueous solution. By doing in this way, it becomes possible to produce the hydrogel base material in which said 2nd cell was embedded in the area | region B thru | or the area | region C. *

In the invention according to this aspect, the flow path structure X is connected to at least one of the inlets G1 to Gn (n ≧ 1) and the inlets G1 to Gn, and is not limited thereto. Pm has inlet channels CG1 to CGn that merge with the merging channel G at any one of Pm, and the gelling agent aqueous solution is continuously introduced into the channel structure X through the inlets G1 to Gn. It is preferable. By doing in this way, it becomes possible to utilize the hydrogel base material which gelatinized inside the flow-path structure and has a more uniform diameter or thickness. *

In the invention according to this aspect, the flow path structure X is connected to at least one of the inlets B1 to Bn (n ≧ 1) and the inlets B1 to Bn, and is not limited thereto. It is preferable to have inlet channels CB1 to CBn that merge with the merged channel G at any one of Pm, and the buffer aqueous solution is continuously introduced into the channel structure X through the inlets B1 to Bn. . By doing in this way, it becomes possible to utilize the hydrogel base material with a more uniform diameter or thickness. *

Further, in the invention according to this aspect, although not limited, the first aqueous solution is at least one of the upper, lower, left, and right sides at at least one of the junctions P1 to Pm. It is preferable to contact with the aqueous solution. By doing in this way, it becomes possible to form the hydrogel base material in which the cross section was comprised by the some area | region more simply. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, the flow-path structure X may be comprised at least partially by the capillary tube. By doing in this way, the hydrogel base material efficiently produced by making said 1st aqueous solution, said 2nd aqueous solution, and said 3rd aqueous solution contact through a capillary tube, and making it gelatinize after that. In addition, it is possible to easily produce a hydrogel substrate having an arbitrary cross-sectional shape by using a capillary tube or a multi-tube arranged in parallel at an arbitrary position. Become. *

Further, in the invention according to this aspect, although not limited, the flow channel structure X may be configured at least partially by a micro flow channel structure manufactured using a microfabrication technique. By doing in this way, it becomes possible to produce the hydrogel base material which has arbitrary cross-sectional shapes and a diameter or thickness of 1 millimeter or less accurately and simply. *

Further, in the invention according to this aspect, although not limited, at least one of the values such as the width, the depth, and the diameter of the flow path structure X is at least partially 1 millimeter or less. It is preferable. In this way, a hydrogel material having a diameter of about 100 to 500 micrometers can be accurately and easily produced. *

Moreover, in the invention which concerns on this viewpoint, although it does not necessarily limit, it is preferable that the density | concentration of the sodium alginate contained in said 1st aqueous solution thru | or said 3rd aqueous solution is 1 g or less per 100 mL, respectively. By doing in this way, the hydrogel base material with the low elasticity modulus of the area | region A and the area | region C can be produced simply and correctly. *

Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable that the density | concentration of the sodium alginate contained in said 2nd aqueous solution is 1 g or more per 100 mL. By doing in this way, it becomes possible to produce the hydrogel base material with the high elasticity modulus of the area | region B simply and correctly. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable that the said gelatinizer aqueous solution contains barium chloride. By doing in this way, compared with the hydrogel base material which contains only calcium ion as a gelling agent, since the swelling of the hydrogel base material when immersed in the culture solution containing a phosphate etc. is suppressed. The embedded cell density can be kept high. *

Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable that the said gelatinizer aqueous solution thru | or the said buffer aqueous solution contain a thickener. By doing so, it becomes possible to stabilize the flow of the laminar flow formed in the flow path, and it becomes possible to produce and use a hydrogel material having a uniform diameter or thickness. *

Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable that said 1st aqueous solution thru | or said 3rd aqueous solution contain propylene glycol alginate. By doing in this way, it becomes possible to produce the hydrogel base material which consists of several parts from which a cross section differs in an elasticity modulus simply and correctly. *

Moreover, in the invention which concerns on this viewpoint, although it does not necessarily limit, it is preferable that the density | concentration of the propylene glycol alginate contained in said 1st aqueous solution thru | or said 3rd aqueous solution is 1 g or more per 100 mL, respectively. By doing so, it becomes possible to produce and use a hydrogel base material in which the elastic modulus of the region A and the region C is sufficiently low. *

The invention according to another aspect of the present invention is a cell evaluation method using the cell evaluation hydrogel substrate according to any one of claims 1 to 32. In such an evaluation method, a plurality of types of cells can be placed in a three-dimensional hydrogel matrix with controlled positions, so that cell infiltration using a planar culture substrate that has been used so far is used. Compared with an evaluation method or the like, it is possible to evaluate cells under conditions closer to the in vivo environment. *

Moreover, in the invention according to this aspect, although not limited, a culture operation is performed on the cells embedded in the hydrogel base material, and the first cells pass inside the hydrogel base material. It is preferable to quantitatively evaluate the infiltration of cells by measuring the number or ratio of infiltration and reaching the outside of the gel. By doing so, it becomes possible to easily, quickly and accurately quantify the proportion of cells that have infiltrated the inside of the hydrogel substrate and proliferated to the outside. It is very effective in evaluating the behavior.

Since the present invention is configured as described above, it is useful for evaluating the invasion of cancer cells, and the cancer cells are arranged at arbitrary positions inside the cross section, and the surroundings are surrounded by other cells. Thus, it is possible to use a fiber-like or sheet-like hydrogel substrate. By culturing the cells in the hydrogel substrate, the culturing operation of cancer cells and cells other than cancer cells in a three-dimensionally close state, which was impossible with the existing cell culture technique, is possible. It becomes possible. *

In addition, since the present invention is configured as described above, the initial position of the cancer cells embedded in the hydrogel substrate can be controlled within a certain range, and the hydrogel group can be controlled. By using a softened portion having a low elastic modulus provided in the cross section of the material and an extracellular matrix component introduced inside, it is possible to promote invasion of cancer cells. Therefore, compared to existing methods and systems, it is possible to evaluate the invasion behavior of cancer cells with higher reproducibility, accurately, simply and efficiently.

Furthermore, since the present invention is configured as described above, it is possible to use a sheet-like or fiber-like hydrogel base material that is accurately and easily produced using a fine channel structure. It becomes. Therefore, when producing a hydrogel base material, the outstanding effect that the complicated apparatus and mechanism for controlling the position of the cell to be embedded is not exhibited is exhibited. *

Furthermore, since the present invention is configured as described above, the infiltration behavior of the cell in an environment closer to the living body using the hydrogel substrate in which the position of the embedded cell is accurately controlled is shown. It is possible to provide an accurate and simple method for evaluation. Therefore, for example, by adding a drug such as an anticancer drug to the culture solution as needed during culture, it becomes possible to perform a drug assay in a state close to the in vivo environment, and as a model for cancer cell research This makes it possible to provide new methods that are useful in the field of drug discovery or in cell physiology research.

BRIEF DESCRIPTION OF THE DRAWINGS It is the schematic which showed an example of the hydrogel base material for fibrous cell evaluation based on embodiment, FIG. 1 (a) is the schematic which showed a part of fiber-like hydrogel in three dimensions, 1 (b) is a schematic view showing a cross section S of the hydrogel substrate for cell evaluation in the plane b in FIG. 1 (a), and FIG. 1 (c) is a cross section in the plane c of the hydrogel substrate for cell evaluation. FIG. It is the schematic which showed typically the mode of the cell before and behind culture | cultivation operation in the hydrogel base material for cell evaluation which embedded two types of cells based on embodiment, FIG.2 (a) is before and after culture | cultivation. FIG. 2B is a schematic diagram showing a cross section of the hydrogel substrate in three dimensions, and FIG. 2B is a schematic diagram showing a cross section of the hydrogel substrate in a plane perpendicular to the longitudinal axis before and after the culture. FIG. It is the schematic which showed the fiber-type or sheet-like hydrogel base material which concerns on embodiment, FIG.3 (a) is an outline of the hydrogel fiber which a cross section is circular and consists of the area | region A and the area | region B. FIG. 3 (b) is a schematic view of a hydrogel fiber having a rectangular cross section and composed of region A, region B, and region C. FIG. 3 (c) has a certain thickness. And it is the schematic of the hydrogel sheet which consists of several area | region A, the area | region C, and the area | region B. It is the schematic diagram which showed a mode that the hydrogel base material for cell evaluation using the flow-path structure based on embodiment was produced, Fig.4 (a) shows the flow-path structure X formed planarly. FIG. 4B is a schematic diagram schematically showing the state observed from the surface and the state of the flow of the solution and cells in the inside, and FIG. 4B is a flow path along the line AA ′ in FIG. FIG. 4C is a schematic diagram showing a cross section of the channel structure X along the line BB ′ in FIG. 4A. The part of the capillary tube in the flow path structure X partly comprised by the capillary tube arrange | positioned in parallel for producing the sheet-like hydrogel base material for cell evaluation based on embodiment is shown typically. The numerals 1, 2, and 3 in the figure are outlets for discharging the first aqueous solution, the second aqueous solution, and the third aqueous solution, respectively. In Example, it is the schematic which showed the microfluidic device which has the flow-path structure X produced by superimposing the four acrylic plates utilized in order to produce the hydrogel base material for fibrous cell evaluation 6 (a) to 6 (d) respectively show a channel structure formed on the lower surface of the first acrylic plate from the top, a channel structure formed on the lower surface of the second acrylic plate, and three FIG. 6 (f) and FIG. 6 are schematic views showing the flow channel structure formed on the upper surface of the acrylic plate of the layer and the flow channel structure formed on the lower surface of the flow channel structure of the third layer. Fig. 6 (c) is a view of the microfluidic device in Fig. 6 (g), and Fig. 6 (e) is an enlarged view of the region e in Fig. 6 (d) and the micro in Fig. 6 (f) and Fig. 6 (g). FIG. 6C is a view of the fluidic device as viewed from the arrow C, 6 (g), respectively, of the microfluidic device, in FIG. 6 (a) ~ FIG 6 (e), A-A 'line, B-B' is a cross-sectional view taken along line. FIG. 7 is a photomicrograph showing a state before and after cell culture in a fiber-shaped hydrogel base material having a cross-section of regions A, B, and C produced using the flow channel structure shown in FIG. 6 in Examples. FIG. 7 (a) is a micrograph of a hydrogel base material in which cancer cells are embedded in region A, and FIG. 7 (b) is a hydrogel base material shown in FIG. 7 (a). FIG. 7 (c) is a photomicrograph after 1 week of cell culture, and FIG. 7 (c) shows the preparation of a hydrogel substrate in which cancer cells are embedded in region A and fibroblasts are embedded in regions B and C. FIG. 7 (d) is a micrograph immediately after cell culture of the hydrogel substrate shown in FIG. 7 (c). In the examples, the infiltration rate of cancer cells when the cisplatin concentration was changed between 0.1 and 100 μM was determined as the fibroblasts in the hydrogel substrate prepared using the flow channel structure shown in FIG. It is the graph which showed the data in each condition at the time of co-culture and when not cultivating cancer cells independently, and the infiltration rate ratio of the vertical axis is in the conditions cultured without administering an anticancer agent The relative ratio of the number of cells infiltrating to the outside of the fiber in each anticancer agent concentration condition with respect to the number of cells infiltrating to the outside of the fiber is shown. In the Examples, cancer cells co-cultured with fibroblasts in a fibrous hydrogel substrate having a cross-section of regions A, B, and C, produced using the flow channel structure shown in FIG. 6, and planar culture FIG. 9A is a graph showing the results of quantitative evaluation of cancer cell-specific gene expression when cisplatin was administered to the treated cancer cells. FIG. 9A shows HIF-1a (hypoxia-inducing factor), FIG. (B) is a graph showing the results of quantifying the gene expression of MMP2 (matrix metalloprotease), and FIG. 9 (c) is the gene expression of VEGF (vascular endothelial growth factor).

Hereinafter, the best mode of the hydrogel substrate for cell evaluation and the cell evaluation method according to the present invention will be described in detail. However, the present invention can be implemented in many different forms, and is not limited only to the embodiments and examples described below. *

FIG. 1 is a schematic view showing an example of a fiber-like hydrogel base material for cell evaluation, and FIG. 1 (a) is a schematic view showing a part of the fiber-like hydrogel in three dimensions. FIG. 1 (b) is a schematic diagram showing a cross section S in the plane b of FIG. 1 (a) of the cell evaluation hydrogel substrate, and FIG. 1 (c) is a plane of the cell evaluation hydrogel substrate. It is sectional drawing in c. In this figure, the cells embedded inside are not drawn. *

The hydrogel base material shown in FIG. 1 is comprised by the area | region A comprised by the area | region A and the area | region C comprised with the hydrogel with a relatively low elasticity modulus, and the hydrogel with a relatively high elasticity modulus. . *

The hydrogel substrate shown in FIG. 1 is formed of a polymer mainly composed of alginic acid. Alginic acid gels quickly in the presence of multivalent cations such as calcium and barium, and there is no need to control the temperature during gelation, solification or dissolution, so cell survival and It is convenient for embedding cells in a hydrogel substrate while maintaining the function. In addition to alginic acid, it is also possible to use a hydrogel substrate formed using a natural or synthetic polymer, such as a polymer formed by polymerization of polyethylene glycol diacrylate, or polyacrylamide. It is also possible to use agarose, collagen, cross-linked gelatin and the like. Furthermore, it is also possible to use a hydrogel substrate formed using a plurality of types of these polymers. *

In addition, in the case of the hydrogel base material formed using alginic acid, it is preferable that the propylene glycol alginate is contained in the hydrogel base material which comprises the area | region A and the area | region C. Propylene glycol alginate is an ester derivative of alginic acid and has a characteristic that it is difficult to gel even in the presence of a polyvalent cation. Therefore, by adding a certain amount of propylene glycol alginate with respect to alginic acid, it is possible to prevent shrinkage of alginic acid generated during gelation during production of the hydrogel base material, and to efficiently reduce the elastic modulus of region A and region C. It is possible. *

FIG. 2 shows a schematic diagram schematically showing the state of cells before and after the culturing operation in the hydrogel base material for cell evaluation in which two types of cells are embedded. FIG. It is the schematic diagram which showed the cross section of the hydrogel base material in three dimensions before and behind culture | cultivation, FIG.2 (b) shows the cross section in the plane orthogonal to the axis | shaft of the length direction of the hydrogel base material before and after culture | cultivation. It is the shown schematic diagram. *

As shown in FIG. 2, the first cell to be evaluated embedded in the region A located near the center of the hydrogel moves toward the outside of the hydrogel substrate by performing a culture operation. And / or grow and infiltrate. At this time, the region A in the hydrogel base material does not need to be in contact with the peripheral edge portion of the cross section S of the hydrogel base material, and thus may not be located near the center portion. *

By using cancer cells as the first cells embedded in the region A, it is possible to evaluate the invasion of cancer cells. For example, screening for searching for new anticancer agents, evaluation of drug efficacy, or cancer Physiological analysis of cells can be performed. As cancer cells, various cultured cell lines can be used, and primary cells can be used as necessary. Further, not only cancer cells but also various stem cells and normal cells with high proliferation ability can be used for evaluation. *

In addition, even if the second cell is not embedded in the region B and the region C in the hydrogel substrate, the infiltration behavior in a three-dimensional environment is evaluated by culturing the first cell alone. It is possible. However, by embedding the second cells in the region B and the region C, it becomes possible to perform invasion evaluation of cancer cells in an environment that more mimics the tissue of a living body. *

As the second cell, any cell such as various cultured cells or primary cells can be used. However, it is preferable to appropriately select the type according to the target cancer cell. Examples of second cells are fibroblasts, endothelial cells, epithelial cells, smooth muscle cells, skeletal muscle cells, mesothelial cells, parenchymal cells, glandular cells, epidermal cells, nerve cells, osteoblasts, of these cells Progenitor cells, cells differentiated from various stem cells, and the like can be used. *

In addition, it is possible to add extracellular matrix molecules such as collagen, fibronectin and laminin, and extracellular matrix mixtures such as matrigel to the matrix constituting the hydrogel base material, and also constitute the hydrogel. As a main component, alginic acid to which a peptide such as cell-adhesive RGD is covalently bonded can also be used. In this way, in addition to mimicking the in vivo environment rich in cell-adhesive matrix, in particular, by introducing these components into regions A and C, cancer cell matrix It is possible to increase the adhesion, increase the infiltration rate, and further reduce the time required for infiltration. *

The overall size of the hydrogel base material may be any extent as long as it allows cancer cell invasion evaluation. However, when the degree of infiltration is evaluated by measuring the proportion of cells that have appeared outside, it is preferable that the diameter or thickness of the hydrogel substrate has a small value. However, when measuring the degree of oxygen deficiency, the second cell embedded in the region B may have a value that is deficient enough to supply oxygen to the cancer cells located in the center. It may be preferable. From these viewpoints, the diameter of the hydrogel is preferably 50 micrometers or more and 500 micrometers or less, and more preferably 50 micrometers or more and 300 micrometers or less.

In addition, the hydrogel base material may be partially non-uniform in diameter or thickness in the length direction, and the region A, the region B, and the region in the cross section may be changed, Moreover, the form which has a branch in the middle may be sufficient, and also the chemical composition of each area | region may differ. However, a hydrogel base material having a uniform cross section and the shape and composition of the region constituting the cross section is more preferable from the viewpoint that the conditions during the culture of the embedded cells can be made uniform. *

The length of the hydrogel substrate is preferably an appropriate length depending on the application. However, in order to perform a highly reliable evaluation experiment, it is preferable that there are 100 or more first cells to be evaluated, and more preferably 300 or more. From such a viewpoint, the length of the hydrogel substrate is preferably 1 millimeter or more, and more preferably 10 millimeters or more. *

The area of the region A in the cross section S is preferably 10D ² or less, where D is the average diameter of the first cells, from the viewpoint of restricting the initial position of the embedded first cells to a certain value.

In addition, the relative position of the region A in the cross-section S is that of the cross-section S from the viewpoint of preventing the difference in culture conditions from occurring due to the difference in the position where the first cells embedded in the region A exist. It is desirable to be at a relatively uniform distance from the periphery. Therefore, when the area of the region A is S and the peripheral length of the region A is L, the circularity 4πS / L ² of the region A is preferably 0.5 or more.

Further, since the width of the region C becomes a value close to the average diameter of the cells, the efficiency of controlling the infiltration direction of the cells is increased. Therefore, if the average diameter of the first cells is D, the region C The width is preferably 3D or less. *

If the density of the first cells embedded in the region A is too low, the efficiency of evaluating the cells decreases. Therefore, the density is preferably higher, and preferably 100,000 or more per cubic centimeter of the hydrogel substrate. However, if the density is too high, cells that are adjacent in the length direction adhere to each other to form a single colony, so that it is also necessary that the density is not more than a certain value. The constant value in this case is, for example, 10 million pieces per cubic centimeter of the hydrogel substrate, or a density such that the average distance between adjacent cells is 100 micrometers or less. *

The density of the second cells embedded in the region B to the region C is preferably higher from the viewpoint of imitating the biological tissue in which the cells are packed at a high density, and is 1 million or more per cubic centimeter of the hydrogel substrate. Preferably there is. Regarding the second cell, cells adjacent in the length direction may adhere to each other to form one colony. *

As shown in FIG. 2, cancer cells infiltrate to the outside by performing an appropriate culture operation. Thereafter, the cells can be evaluated by quantitatively evaluating the degree of invasion of cancer cells in a three-dimensional culture environment as necessary. The simplest means for quantifying the degree of infiltration is to observe the cells that have infiltrated to the outside using a microscope or the like and simply measure the ratio. In addition, evaluation of survival rate by staining, etc., quantification of infiltration in hydrogel matrix using image analysis technology, quantification of gene expression by real-time PCR, etc. may be performed independently or simultaneously. Is possible. *

As a cell culturing operation, it is preferable to immerse the produced hydrogel base material in a culture solution and maintain it under controlled temperature, oxygen concentration, and carbon dioxide concentration conditions. As a culture solution used in the culturing operation, a general culture solution for cell culture according to the target cell type can be used. Moreover, at the time of culture | cultivation, it is preferable to immerse the hydrogel base material in the culture solution with which the petri dish, the flask, etc. were filled, and culture | cultivate using culture apparatuses, such as a CO2 incubator. Furthermore, a shaking operation can be performed as necessary. *

For example, when evaluating the effect of drugs on cell invasion, the drug concentration and the degree of invasion can be easily quantitatively evaluated by adding the drug to the culture medium and culturing. Is possible. In addition, after immersing the hydrogel substrate in a solution containing the drug for a certain period of time, culturing it in a culture solution that does not contain the drug allows the drug to act for a certain period of time and evaluate its effect. It is. *

In addition, as a hydrogel base material, it is possible to use the thing of various shapes other than what illustrated the schematic to FIG. 1 and FIG. *

FIG. 3 is a schematic view showing an example of a fiber-like or sheet-like hydrogel substrate. FIG. 3A shows a hydrogel having a circular cross section and comprising regions A and B. Fig. 3 (b) is a schematic view of a fiber, and Fig. 3 (b) is a schematic view of a hydrogel fiber having a rectangular cross section and composed of a region A, a region B, and a region C. Fig. 3 (c) It is the schematic of the hydrogen sheet which has thickness and consists of the several area | region A, the area | region C, and the area | region B. FIG. *

By using the hydrogel substrate as shown in FIG. 3 (a), the first cell embedded in the region A is not the outside of the hydrogel substrate, but the inside of the region A is the length of the hydrogel substrate. It is possible to observe the infiltration in the direction. Further, in the case of a hydrogel base material composed of three regions of region A, region B, and region C as shown in FIG. 1, the cross section may be rectangular as shown in FIG. Furthermore, as shown in FIG.3 (c), it is a sheet-like hydrogel base material, Comprising: You may have several area | region A containing a 1st cell in the inside. In particular, in the case of the sheet-like hydrogel substrate shown in FIG. 3 (c), it becomes possible to embed first cells to be evaluated in high density and high efficiency in a planar form. It becomes possible to evaluate cells more efficiently. *

As a process for producing these hydrogel substrates, those using a microchannel structure are very efficient. *

FIG. 4 is a schematic diagram showing a state in which a hydrogel substrate for cell evaluation using a channel structure is produced. FIG. 4A shows a channel structure formed in a plane. FIG. 4B is a schematic diagram schematically showing the state of X observed from the surface and the state of the flow of the solution and cells in the inside, and FIG. 4B is the AA ′ line in FIG. FIG. 4C is a schematic view showing a cross section of the flow path structure X along the line BB ′ in FIG. 4A. *

The flow channel structure X shown in FIG. 4 has eight inlet channels I1, I2, I2 ′, I3, B1, B1 ′, G1, G1 ′, which are connected to the inlet channels CI1, CI2, CI2 ′, CI3, CB1, respectively. CB 1 ′, CG 1, CG 1 ′, merging points P 1, P 2, P 3, P 4 where the inlet channels merge in stages, and a merging channel G existing between the merging point P 4 and the outlet O are included. . *

In the case of producing a planar flow path structure as shown in FIG. 4, the material of the device is PDMS (polydimethylsiloxane), various polymer materials such as acrylic, glass, silicon, ceramics, stainless steel, etc. These various metals can be used, and any of these materials can be used in combination. However, in order to produce a planar flow path at low cost, it is preferable to use a polymer material at least partially. As a processing technology for the channel structure, a manufacturing technique using a mold such as molding or embossing is preferable in that the channel structure can be easily manufactured. In addition, wet etching, dry etching, laser It is also possible to use manufacturing techniques such as processing, electron beam direct drawing, and machining. *

The flow channel structure shown in FIG. 4 needs to be designed to have an appropriate inner diameter, width, and depth according to the size of the hydrogel substrate to be produced. However, in order to efficiently produce a hydrogel substrate having a diameter or thickness of 500 micrometers or less, any value such as the diameter, depth, or width of the channel structure should be 1 millimeter or less. Is preferable, and it is more preferable that it is 500 micrometers or less. *

The first aqueous solution, the second aqueous solution, and the third aqueous solution, each of which is a sol aqueous solution for constituting a hydrogel and in which cells are suspended, are respectively input to the channel structure X shown in FIG. Introduce continuously through I1, inlets I2 and I2 ', inlet I3. Further, the gelling agent aqueous solution is continuously introduced from the inlet G1 and the inlet G1 ', and the buffer aqueous solution is continuously introduced from the inlet B1 and the inlet B1', respectively. *

By appropriately adjusting the introduction flow rate at the junction P4, the first aqueous solution, the second aqueous solution, and the third aqueous solution form a laminar flow in a state where they are in contact with each other. And in the confluence | merging flow path G in the downstream, these gelatinous solutions will gelatinize gradually from the outside, and a fiber-like hydrogel base material is continuously formed from the exit O. . *

By devising the three-dimensional arrangement of the inlet channel structure, the three-dimensional structure of the channel at the branch points P1 and P2, and the respective introduction flow rates, the first aqueous solution, the second aqueous solution, It is possible for the three aqueous solutions to have any arrangement, and by gelling these solutions while maintaining the arrangement, it is possible to obtain a hydrogel substrate that retains the pattern. is there. For example, by forming a cross-sectional pattern as shown in FIG. 4B in the confluence channel G, a hydrogel substrate as shown in FIGS. 1 and 2 can be produced. *

As the gelling agent contained in the gelling agent aqueous solution, any polyvalent metal cation capable of gelling alginic acid can be used. However, from the viewpoint of cytotoxicity, the ions are preferably divalent cations of calcium, strontium, barium, magnesium, or any mixture thereof. Moreover, since these ions need to be water-soluble, the gelling agent aqueous solution is preferably an aqueous solution in which those chlorides are dissolved. Especially, it is more preferable that gelling agent aqueous solution contains barium chloride from a viewpoint of suppressing the swelling in the culture solution of the produced hydrogel material, and keeping the cell density in a hydrogel base material high. In order to reduce damage to cells, it is desirable that the osmotic pressure of the aqueous gelling agent solution is adjusted to an optimal value in advance. *

Since the flow channel structure X shown in FIG. 4 performs gelation in the inside thereof, it is designed so that an aqueous gelling agent solution can be introduced from the middle of the flow channel. In order to prevent clogging of the flow path due to gelation, it is designed so that an aqueous buffer solution containing no gelling agent can be introduced. However, the gel may be formed outside the channel by not introducing these aqueous solutions, closing the inlets B1, B2 ', G1, G1' and immersing the outlet O in the gelling agent solution. However, from the viewpoint that it is easy to accurately adjust the diameter of the obtained fiber-like hydrogel substrate, a system in which gelation is performed inside the flow path is preferable. *

Moreover, since the aqueous solution containing sodium alginate generally has high viscosity, it is preferable that a thickener is added to the gelling agent aqueous solution and / or the buffer aqueous solution in advance. As the thickener, dextran, polyethylene glycol, polymethylcellulose, propylene glycol alginate, or any combination thereof can be used. By adding these thickeners to an aqueous gelling agent solution or an aqueous buffer solution, it becomes possible to stably form a laminar flow when gelling in a flow path as shown in FIG. It is possible to improve the operability when producing the gel material. Of the first aqueous solution, the second aqueous solution, the third aqueous solution, the gelling agent aqueous solution, and the buffer aqueous solution, the ratio of the viscosity of the smallest to the largest at room temperature is 1: 1 to 1: It is preferably in the range of 100, more preferably in the range of 1: 1 to 1:10. Note that this is not the case when gelation is performed outside the flow path.

In addition to the planar flow channel structure shown in FIG. 4, it is also possible to use a flow channel structure in which capillary tubes are arranged in parallel or multiplexed. *

FIG. 5 schematically shows a portion of the capillary tube in the flow channel structure X partially formed by the capillary tubes arranged in parallel for producing a sheet-like hydrogel substrate for cell evaluation. The numerals 1, 2, and 3 in the figure are outlets for discharging the first aqueous solution, the second aqueous solution, and the third aqueous solution, respectively. By using such a paralleled capillary structure, the first aqueous solution, the second aqueous solution, and the third aqueous solution are merged, and then continuously discharged into the gelling agent aqueous solution. It is also possible to produce a sheet-like hydrogel structure as shown in FIG. *

The alginic acid contained in the first aqueous solution, the second aqueous solution, and the third aqueous solution introduced into the flow channel structure as described above is generally a sodium salt. Any alginic acid having any molecular weight may be used as long as it forms a hydrogel in the presence of a polyvalent cation. However, from the viewpoint of operability during preparation of the hydrogel, it is preferred that the viscosity when 1 g of alginic acid is dissolved in 100 mL of water and kept at room temperature is in the range of 10 cP to 400 cP. In addition, if the strength of the prepared gel is above a certain level, the degree of swelling when the prepared hydrogel material is immersed in the culture solution is low, which is convenient for embedding cells at high density. Of the uronic acids that are units constituting the alginic acid polymer, it is preferable to use those having a guluronic acid ratio of 60% or more. *

The concentration of sodium alginate contained in the first aqueous solution and the third aqueous solution is preferably 1 g or less per 100 mL of each solution in order to keep the elastic modulus of the hydrogel forming the regions A and C low. Furthermore, it is more preferable that it is 0.5 g or less. Further, the concentration of propylene glycol alginate contained in these aqueous solutions is preferably 1 g or more per 100 mL of each solution in order to reduce the elastic modulus of the hydrogel forming the regions A and C. *

On the other hand, the concentration of sodium alginate contained in the second aqueous solution is preferably 1 g or more per 100 mL of the solution in order to increase the elastic modulus of the hydrogel forming the region B. The second aqueous solution preferably does not contain propylene glycol alginate.

Hereinafter, the effect of this invention was confirmed by producing the hydrogel base material for cell evaluation which concerns on the said embodiment, and actually performing cell evaluation. This will be described below. *

FIG. 6 is a schematic diagram showing a microfluidic device having a flow channel structure X, which is produced by superposing four acrylic plates, which is used for producing a fiber-like hydrogel substrate for cell evaluation. 6 (a) to 6 (d) show the flow path structure formed on the lower surface of the first acrylic plate from the top, and the flow formed on the lower surface of the second acrylic plate, respectively. FIG. 6 (f) is a schematic diagram showing a channel structure, a channel structure formed on the upper surface of the third layer acrylic plate, and a channel structure formed on the lower surface of the third layer channel structure, and FIG. ) And FIG. 6 (g) are C arrow views of the microfluidic device, and FIG. 6 (e) is an enlarged view of the region e in FIG. 6 (d), and FIG. 6 (f) and FIG. FIG. 6C is a view of the microfluidic device in FIG. Shown in FIG. 6 (g), respectively, of the microfluidic device, in FIG. 6 (a) ~ FIG 6 (e), A-A 'line, B-B' is a cross-sectional view taken along line. *

The flow path structure shown in FIG. 6 is composed of three 2 mm-thick acrylic flat plates that have been cut using a microfabrication technique and two unmilled acrylic flat plates that are not cut. It is formed by laminating by pressure bonding. *

The channel width in each part of the channel structure X is, for example, the width and depth of the channel existing on the lower surface of the first flat plate is 100 to 500 micrometers. There are 5mm, 3.5mm, and 1.5mm wide channels on the lower surface of the second flat plate, and the depths are 900, 1.2, and 1.5mm, respectively. there were. The width and depth of the channel existing on the upper surface of the third-layer flat plate was 500 micrometers. The width and depth of the channel existing on the lower surface of the third layer flat plate were 100 to 500 micrometers. Of these values, the diameter of the obtained fiber-like hydrogel substrate is controlled by the diameter of the flow path existing on the lower surface of the first flat plate, so that it is thicker or thinner as necessary. It is preferable to use a channel structure. *

As cancer cells, human alveolar basal epithelial adenocarcinoma cell A549 was used, and as cells other than cancer cells, Chinese hamster-derived lung fibroblast cell line V79-379A cells were used. These cells were prepared by culturing in advance on a normal cell culture dish to grow a certain amount, detaching from the plate by enzyme treatment, and removing the culture solution by centrifugation. *

As the first aqueous solution and the third aqueous solution, an aqueous solution in which 0.5 g of propylene glycol alginate, 0.1 g of RGD alginic acid, 0.45 g of sodium chloride, and 5 mM of HEPES was dissolved in 50 mL of water, respectively. , About 30 million A549 cells per mL and about 200 million V79-379A cells per mL were suspended. Of these components, RGD alginate was used for maintaining cell function and promoting infiltration, HEPES for pH adjustment, and sodium chloride for osmotic pressure adjustment. It is also possible to add these components or replace them with other components. *

As the second aqueous solution, an aqueous solution in which 0.5 g of sodium alginate, 0.1 g of RGD alginic acid, 0.45 g of sodium chloride, and 5 mM of HEPES were dissolved in 50 mL of water was used, and V79-379A cells were used. About 200 million cells were suspended per mL. *

Moreover, as gelling agent aqueous solution, barium chloride is 20 mM as a gelling agent, dextran having a molecular weight of 500,000 is 10% (w / v) as a thickening agent, sodium chloride for adjusting osmotic pressure is 120 mM, pH adjustment Therefore, an aqueous solution containing 10 mM HEPES was used. *

As the buffer aqueous solution, an aqueous solution containing 10% (w / v) dextran having a molecular weight of 500,000 as a thickener, 150 mM sodium chloride for adjusting osmotic pressure, and 10 mM HEPES for adjusting pH was used. *

All of these solutions were sterilized by heating or filtering in advance. *

These aqueous solutions were continuously introduced into the channel structure X using a syringe pump. In addition, in order to connect each inlet in the syringe and the flow path structure X, a PTFE tube was used. *

The flow rate of the aqueous solution introduced from each inlet was changed according to the size of the cell evaluation hydrogel base material to be produced. As for the introduction flow rate from each inlet, when the width of the confluence channel G is 300 micrometers and the depth is 300 micrometers, the first aqueous solution introduced from the inlet I1 is 5 to 10 microliters per minute, and the inlet I2 , I2 ′ and I2 ″ were introduced into the second aqueous solution at 10 to 50 microliters per minute, and the third aqueous solution introduced from the inlet I3 was introduced at 5 to 20 microliters per minute and gelled from the inlet G1. The aqueous agent solution was 50 to 300 microliters per minute, and the buffer aqueous solution introduced from the inlet B1 was 5 to 20 microliters per minute. *

The first aqueous solution introduced from the inlet I1 passes through the inlet channel CI1 and is introduced into the vertically upward channel structure formed on the lower surface of the third layer flat plate. At the confluence point P1 existing on the upper surface, it merged with the third aqueous solution introduced from the inlet I3 and passed through the inlet channel CI3. Furthermore, the second aqueous solution introduced from the inlets I2, I2 ′, and I2 ″ merges at the merge point P2, and the buffer aqueous solution and the downstream merge point at the merge point P3 existing on the lower surface of the first flat plate. In P4, the gelling agent aqueous solution was merged with each other. And the flow which the 1st aqueous solution, the 2nd aqueous solution, and the 3rd aqueous solution merged inside the confluence | merging flow path G gelatinizes, and a fiber-like hydrogel base material is formed continuously, and it collects from the exit O It was done. *

FIG. 7 is a photomicrograph showing the state before and after cell culture in a fiber-shaped hydrogel substrate having a cross-section of regions A, B, and C produced using the flow channel structure shown in FIG. FIG. 7 (a) is a photomicrograph of a hydrogel base material in which cancer cells are embedded in region A immediately after production, and FIG. 7 (b) is shown in FIG. 7 (a). FIG. 7 (c) shows a hydrogel with a cancer cell embedded in region A and fibroblasts embedded in region B and region C. FIG. FIG. 7 (d) is a photomicrograph of the hydrogel substrate shown in FIG. 7 (c) after one week of cell culture. *

In the hydrogel substrate shown in FIG. 7, the flow rate of the first aqueous solution introduced from the inlet I1 is 3 microliters per minute, and the flow rate of the second aqueous solution introduced from the inlets I2, I2 ′, and I2 ″ is the total. 72 microliters per minute, the flow rate of the third aqueous solution introduced from the inlet I3 is 15 microliters per minute, the flow rate of the buffer aqueous solution introduced from the inlet B1 is 25 microliters per minute, the gelling agent aqueous solution introduced from the inlet G1 This was obtained when the flow rate was 100 microliters per minute. As shown in FIG. 7 (a), a hydrogel substrate having an average diameter of about 120 micrometers is obtained, and A549 cells, which are cancer cells, are arranged at intervals in the length direction in the center A of the region. It was observed that In the case of a hydrogel substrate prepared by suspending V79-379A cells, which are lung fibroblasts, in the second aqueous solution and the third aqueous solution, the V79-379A cells are densely surrounded around the A549 cells. The arrangement was confirmed. *

In addition, as shown in FIG. 7, when culture was performed for 1 week using a culture solution in a CO ₂ concentration 5% atmosphere, any condition when the lung fibroblasts were embedded or not embedded However, it was confirmed that the center cancer cells infiltrated the hydrogel portion having a low elastic modulus corresponding to the region C to the outside and formed colonies outside the hydrogel substrate as shown in FIG.

The cancer cell invasion behavior was evaluated by measuring the number of cancer cells in the hydrogel that infiltrated the gel embedded with fibroblasts and jumped out of the gel using a microscopic image. It was possible to easily evaluate the infiltration ratio of cancer cells. *

Moreover, the hydrogel base material which embedded the cancer cell in the area | region A in the cross section and the hydrogel base material which embedded the cancer cell in the area | region A and the fibroblast in the area | region B and the area | region C were produced. After culturing the cells for 5 days, the cells were added to a cisplatin medium as an anticancer agent and cultured for 24 hours, and then the cells were further cultured for 24 hours using a medium not containing cisplatin. Thereafter, in order to quantitatively evaluate the effect of the anticancer agent on the invasion behavior of cancer cells, the ratio of cancer cells infiltrating outside the fiber to the number of embedded cancer cells was counted for a total of 300 cancer cells, The infiltration rate was calculated. In addition, single culture was performed without embedding fibroblasts in region B and region C, and comparison with co-culture was performed. *

FIG. 8 shows the infiltration ratio of cancer cells when the cisplatin concentration is changed between 0.1 and 100 μM and the fibroblasts inside the hydrogel substrate prepared using the flow channel structure shown in FIG. It is the graph which showed the data in each condition at the time of co-culture and when not cultivating cancer cells independently, and the infiltration rate ratio of the vertical axis is in the conditions cultured without administering an anticancer agent The relative ratio of the number of cells infiltrating to the outside of the fiber in each anticancer agent concentration condition with respect to the number of cells invading to the outside of the fiber is shown. Each data shows the mean value ± standard deviation of the values obtained by counting 300 cells for four independent samples. *

As shown in FIG. 8, the degree of infiltration of cancer cells varies depending on the concentration of the anticancer agent, and the case where the cancer cells are cultured alone inside the hydrogel substrate, and the case where the cancer cells and fibroblasts are co-cultured And the infiltration behavior was significantly different. In particular, in the system in which two types of cells were co-cultured, the invasion ratio was greatly reduced when the anticancer drug concentration was 10 ⁻⁵ M or more, that is, the anticancer drug worked effectively, whereas the cancer cells were cultured alone. In the system, the concentration was 10 ⁻⁶ M or more, and a large difference was observed. From these results, it was observed that the medicinal effects of anticancer agents differ greatly between co-culture and single culture.

In addition, in order to evaluate the function of cancer cells, cancer cells co-cultured with fibroblasts inside the hydrogel substrate and cancer cells planarly cultured using a normal cell culture plate were prepared. Cisplatin was administered as an anticancer agent under the same conditions. Furthermore, after mRNA was extracted and cDNA was synthesized, gene expression was quantitatively evaluated by real-time PCR using GAPDH as a housekeeping gene and TaqMan probe as a fluorescent probe. In addition, as a target, three types of genes, HIF-1a (hypoxia-inducing factor), MMP2 (matrix metalloprotease), and VEGF (vascular endothelial growth factor) were quantitatively evaluated.

FIG. 9 shows a cancer cell co-cultured with fibroblasts inside a fibrous hydrogel substrate having a cross-section of regions A, B, and C, and a plane produced using the flow channel structure shown in FIG. A graph showing the results of quantitative evaluation of cancer cell-specific gene expression when cisplatin was administered to cultured cancer cells is shown. FIG. 9 (a) shows HIF-1a (hypoxia-inducing factor). 9 (b) is a graph showing the results of quantifying the gene expression of MMP2 (matrix metalloprotease) and FIG. 9 (c) is the VEGF (vascular endothelial growth factor) gene expression. The relative gene expression level on the vertical axis is a relative value based on each gene expression level when cisplatin is not administered in planar culture.

As shown in FIG. 9, it was confirmed that all the gene expression levels were greatly decreased in the cancer cells cultured in a plane, under the condition that the cisplatin concentration was 100 μM. On the other hand, in cancer cells co-cultured with fibroblasts inside the hydrogel substrate, it was confirmed that the expression level of these genes was active without significant decrease even when 100 μM of the anticancer agent was administered. It was. This is considered to be due to imitation of a three-dimensional environment and an environment close to the living body in which normal cells exist around cancer cells. From this result, the three-dimensional proposed in the present invention is also considered. The superiority of the co-cell culture environment and the similarity to the living body were demonstrated.

Since the present invention is configured as described above, a cell culture system using a flat culture substrate, which is usually used in drug discovery screening, drug metabolism / toxicity evaluation tests using cells, etc. In comparison, it is possible to easily and accurately evaluate the infiltration behavior of cells in a three-dimensional environment imitating a living body. Although normal planar culture has the advantage of simplifying operations and equipment, the environment is very different from the normal biological environment for cells, so the environment in the living organism is accurate when performing assays using cells. It is hard to say that it has been reproduced. Therefore, the three-dimensional hydrogel substrate according to the present invention is used not only in the pharmaceutical industry but also in the field of cell biochemical research as a tool for performing a cell assay simply and accurately in an environment reflecting biological structures. It can be an important and versatile new tool that is widely used. *

In addition, since the present invention is configured as described above, it is possible to provide a hydrogel substrate in which cells to be evaluated are accurately arranged at specific positions in a three-dimensional culture environment. It becomes possible. By controlling the initial position of cells, when evaluating the invasion behavior, there is less data variation and higher reliability, so it can be widely used as a system that enables more accurate cell evaluation. Expected. *

Furthermore, since the present invention is configured as described above, it can usually provide a technique for performing a simpler assay using cells that is very time-consuming and less reliable. In particular, simply counting the number of cells that have infiltrated the outside of the hydrogel substrate makes it possible to easily measure the infiltration rate, thus providing a system that does not require complex image analysis techniques or optical systems. Probability is high. Therefore, in terms of cost, it is considered to be superior to existing systems and can provide a novel cell evaluation method widely used in industrial fields and academic fields in which evaluation is performed using cells.

Claims (34)

1. It is composed of hydrogel, has a fiber-like or sheet-like form, and the peripheral edge is not in contact with the peripheral edge of the cross-section S inside the cross-section S in a plane perpendicular to the longitudinal axis; The cell A has a region A in which the first cells exist, and the region A is a hydrogel for cell evaluation that is configured by a hydrogel having a lower elastic modulus than the region B other than the region A in the cross section S. Gel substrate.
2. The hydrogel base material has a region C partially in contact with the region A and the region B in the cross section S and partially in contact with the peripheral edge of the cross section S. The hydrogel substrate for cell evaluation according to claim 1, which is composed of a hydrogel having a lower elastic modulus than that of the region B.
3. The hydrogel base material for cell evaluation according to claim 1, wherein a second cell different from the first cell is present in the region B.
4. The hydrogel substrate for cell evaluation according to claim 2, wherein the second cell is present in the region C.
5. The hydrogel substrate for cell evaluation according to claim 1, wherein at least one of the hydrogels constituting the region A, the region B, and the region C is composed of alginic acid or a derivative of alginic acid.
6. The hydrogel substrate for cell evaluation according to claim 1, wherein propylene glycol alginate is contained in at least one of the hydrogels constituting the region A and the region C.
7. The hydrogel matrix that constitutes the region A, the region B, and the region C contains at least one of a protein that constitutes an extracellular matrix and a cell adhesive peptide molecule. Hydrogel substrate for cell evaluation.
8. The hydrogel substrate for cell evaluation according to claim 1, wherein the first cell is a cancer cell.
9. The hydrogel substrate for cell evaluation according to claim 1, wherein the second cell is a cell other than a cancer cell.
10. The second cells are fibroblasts, endothelial cells, epithelial cells, smooth muscle cells, skeletal muscle cells, mesothelial cells, parenchymal cells, glandular cells, epidermal cells, nerve cells, osteoblasts, and precursors of these cells The hydrogel substrate for cell evaluation according to claim 1, which is at least one of a cell and a cell differentiated from various stem cells.
11. The area of the region A, when the average diameter of the first cell is D, the cell evaluation hydrogel substrate according to claim 1 is 10D ² or less.
12. The hydrogel substrate for cell evaluation according to claim 1, wherein when the area of the region A is S and the peripheral length of the region A is L, the circularity 4πS / L ² of the region A is 0.5 or more. .
13. The hydrogel substrate for cell evaluation according to claim 1, wherein the width of the region C is 3D or less, where D is the average diameter of the first cells.
14. The hydrogel substrate for cell evaluation according to claim 1, which is in a fiber form having a diameter of 500 μm or less.
15. The hydrogel base material for cell evaluation according to claim 1, wherein the hydrogel base material is in the form of a sheet having a thickness of 500 micrometers or less, and a plurality of the regions A exist inside the cross section S.
16. The hydrogel substrate for cell evaluation according to claim 1, wherein the density of the first cells contained in the region A is 100,000 or more per cubic centimeter.
17. The hydrogel substrate for cell evaluation according to claim 1, wherein the density of the second cells contained in the region B and the region C is 1 million or more per cubic centimeter.
18. At least two inlets I1 to In (n ≧ 2), inlet channels CI1 to CIn connected to the inlets I1 to In, respectively, and at least one merging of the inlet channels CI1 to CIn simultaneously or stepwise For a channel structure X having points P1 to Pm (m ≧ 1), a merging channel G existing downstream from the merging points P1 to Pm, and an outlet O existing downstream from the merging channel G, A first aqueous solution containing sodium alginate and in which the first cells are suspended is passed through the inlet I1, and a second aqueous solution containing sodium alginate is passed through the inlet I2, respectively. The first aqueous solution and the second aqueous solution are brought into contact with each other inside the flow channel structure X, and the first aqueous solution and the front solution are introduced inside or outside the flow channel structure X. The second aqueous solution is produced by bringing the first aqueous solution and the second aqueous solution into contact with each other by bringing the second aqueous solution into contact with the aqueous gelling agent solution, and the region. The cell according to claim 1, wherein A is formed by gelation of the first aqueous solution, and the region B is formed by gelation of the second aqueous solution. Hydrogel substrate for evaluation.
19. At least three inlets I1 to In (n ≧ 3), inlet channels CI1 to CIn connected to the inlets I1 to In, respectively, and at least one junction in which the inlet channels CI1 to CIn merge simultaneously or stepwise For a channel structure X having points P1 to Pm (m ≧ 1), a merging channel G existing downstream from the merging points P1 to Pm, and an outlet O existing downstream from the merging channel G, A first aqueous solution containing sodium alginate and in which the first cells are suspended is added via the inlet I1, and a second aqueous solution containing sodium alginate is added via the inlet I2. The third aqueous solution containing sodium alginate is continuously introduced through the inlet I3, and the first aqueous solution, the second aqueous solution, the third aqueous solution are introduced into the flow path structure X. Contacting the solution, and further bringing the first aqueous solution, the second aqueous solution, and the third aqueous solution into contact with the gelling agent aqueous solution inside or outside the flow path structure X, The region A is formed by gelation of the first aqueous solution, and the region A is formed by continuous gelation in a state where the second aqueous solution and the second aqueous solution are in contact with each other. And the region B is formed by gelation of the second aqueous solution, and the region C is formed by gelation of the third aqueous solution. Item 2. The hydrogel substrate for cell evaluation according to Item 1.
20. The hydrogel substrate for cell evaluation according to claim 18, wherein the second cells are suspended in at least one of the second aqueous solution and the third aqueous solution.
21. The flow path structure X includes at least one inlet G1 to Gn (n ≧ 1) and an inlet flow path CG1 that is connected to the inlets G1 to Gn and merges with the merge flow path G at any of the merge points P1 to Pm. 19. The hydrogel substrate for cell evaluation according to claim 18, wherein the gelling agent aqueous solution is continuously introduced into the channel structure X through the inlets G1 to Gn.
22. The flow path structure X includes at least one inlet B1 to Bn (n ≧ 1) and an inlet flow path CB1 that is connected to the inlets B1 to Bn and merges with the merge flow path G at any of the merge points P1 to Pm. The hydrogel substrate for cell evaluation according to claim 18, wherein the aqueous buffer solution is continuously introduced into the channel structure X through the inlets B1 to Bn.
23. The hydrogel group for cell evaluation according to claim 18, wherein the first aqueous solution is in contact with the second aqueous solution in at least one of the upper, lower, left and right directions at at least one of the junctions P1 to Pm. Wood.
24. The hydrogel substrate for cell evaluation according to claim 18, wherein the flow channel structure X is at least partially configured by a capillary tube.
25. The hydrogel substrate for cell evaluation according to claim 18, wherein the flow channel structure X is at least partially configured by a micro flow channel structure manufactured by using a microfabrication technique.
26. The hydrogel substrate for cell evaluation according to claim 18, wherein at least one of the values of the width, depth, and diameter of the channel structure X is at least partially 1 millimeter or less.
27. The hydrogel substrate for cell evaluation according to claim 18, wherein the concentration of sodium alginate contained in at least one of the first aqueous solution, the second aqueous solution, and the third aqueous solution is 1 g or less per 100 mL. .
28. The hydrogel substrate for cell evaluation according to claim 18, wherein the concentration of sodium alginate contained in the second aqueous solution is 1 g or more per 100 mL.
29. The hydrogel substrate for cell evaluation according to claim 18, wherein the gelling agent aqueous solution contains barium chloride.
30. The hydrogel substrate for cell evaluation according to claim 18, wherein the aqueous gelling agent solution or the aqueous buffer solution contains a thickener.
31. The hydrogel substrate for cell evaluation according to claim 18, wherein the first aqueous solution to the third aqueous solution contain propylene glycol alginate.
32. The hydrogel substrate for cell evaluation according to claim 31, wherein the concentration of the propylene glycol alginate contained in the first aqueous solution to the third aqueous solution is 1 g or more per 100 mL.
33. A cell evaluation method using the hydrogel substrate for cell evaluation according to claim 1.
34. For the cells embedded in the hydrogel base material, a culture operation is performed, and the number or ratio of the first cells reaching the outside of the gel by infiltrating the inside of the hydrogel base material, The cell evaluation method according to claim 33, which quantitatively evaluates cell infiltration.

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