WO2014126228A1 - Hydrogel substrate for cell evaluation, and cell evaluation method - Google Patents
Hydrogel substrate for cell evaluation, and cell evaluation method Download PDFInfo
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- WO2014126228A1 WO2014126228A1 PCT/JP2014/053578 JP2014053578W WO2014126228A1 WO 2014126228 A1 WO2014126228 A1 WO 2014126228A1 JP 2014053578 W JP2014053578 W JP 2014053578W WO 2014126228 A1 WO2014126228 A1 WO 2014126228A1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/74—Alginate
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2535/00—Supports or coatings for cell culture characterised by topography
Definitions
- the present invention relates to a hydrogel substrate for cell evaluation and a cell evaluation method.
- 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.
- 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.
- 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.
- 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.
- spherical aggregates of cancer cells spheroids
- Patent Document 1 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.
- Patent Document 2 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.
- Non-Patent Document 1 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. *
- 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.
- 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. *
- 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.
- 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.
- 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.
- the first cell to be evaluated such as a cancer cell
- 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.
- 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.
- region B it is desirable for the area
- region C it is desirable for the area
- region C is comprised with the derivative of alginic acid or alginic acid.
- region C contains propylene glycol alginate.
- 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. *
- said 1st cell is a cancer cell.
- said 2nd cell is cells other than a cancer cell.
- 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.
- 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.
- the circularity 4 ⁇ S / L 2 of the region A is 0.5 or more.
- 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.
- region C is 3D or less.
- 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.
- 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. *
- region A is 100,000 or more per 1 cubic centimeter.
- the density of the second cells included in the regions B to C is preferably 1 million or more per cubic centimeter.
- 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
- region B is formed by gelation of the second aqueous solution.
- 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.
- aqueous solution 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
- said 2nd cell is suspended in said 2nd aqueous solution thru
- 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.
- 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. *
- 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
- the flow-path structure X may be comprised at least partially by the capillary tube.
- 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.
- 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.
- 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.
- or said 3rd aqueous solution is 1 g or less per 100 mL, respectively.
- concentration of the sodium alginate contained in said 2nd aqueous solution is 1 g or more per 100 mL.
- the said gelatinizer aqueous solution contains barium chloride.
- 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.
- or said 3rd aqueous solution contain propylene glycol alginate.
- or said 3rd aqueous solution is 1 g or more per 100 mL, respectively.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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. *
- 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.
- a drug such as an anticancer drug
- 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)
- 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
- FIG.2 (a) is before and after culture
- FIG. 2B is a schematic diagram showing a cross section of the hydrogel substrate in three dimensions
- 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
- 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.
- 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.
- 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.
- 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)
- 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
- 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
- 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).
- the infiltration rate of cancer cells when the cisplatin concentration was changed between 0.1 and 100 ⁇ M 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.
- 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)
- FIG. 9 (c) is the gene expression of VEGF (vascular endothelial growth factor).
- FIG. 1 is a schematic view showing an example of a fiber-like hydrogel base material for cell evaluation
- 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
- 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
- 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.
- 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.
- a hydrogel substrate formed using a plurality of types of these polymers are also possible to use. *
- the propylene glycol alginate is contained in the hydrogel base material which comprises the area
- 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
- 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.
- 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.
- 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.
- 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. *
- 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.
- 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.
- 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.
- extracellular matrix molecules such as collagen, fibronectin and laminin, and extracellular matrix mixtures such as matrigel
- extracellular matrix molecules such as collagen, fibronectin and laminin, and extracellular matrix mixtures such as matrigel
- extracellular matrix molecules such as collagen, fibronectin and laminin, and extracellular matrix mixtures such as matrigel
- alginic acid to which a peptide such as cell-adhesive RGD is covalently bonded can also be used.
- alginic acid to which a peptide such as cell-adhesive RGD is covalently bonded can also be used.
- 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.
- 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.
- 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.
- 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 4 ⁇ S / L 2 of the region A is preferably 0.5 or more.
- 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.
- 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.
- the second cell cells adjacent in the length direction may adhere to each other to form one colony.
- 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.
- 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. *
- 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.
- a culture solution used in the culturing operation a general culture solution for cell culture according to the target cell type can be used.
- 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
- 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.
- 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. *
- hydrogel base material 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
- 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.
- 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.
- the cross section may be rectangular as shown in FIG.
- FIG.3 (c) it is a sheet-like hydrogel base material, Comprising: You may have several area
- 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. *
- 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. . *
- 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.
- 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.
- 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.
- 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.
- 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 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.
- 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
- the gelling agent contained in the gelling agent aqueous solution any polyvalent metal cation capable of gelling alginic acid can be used.
- the ions are preferably divalent cations of calcium, strontium, barium, magnesium, or any mixture thereof.
- the gelling agent aqueous solution is preferably an aqueous solution in which those chlorides are dissolved.
- 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.
- the osmotic pressure of the aqueous gelling agent solution is adjusted to an optimal value in advance.
- 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.
- 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.
- a system in which gelation is performed inside the flow path is preferable. *
- the aqueous solution containing sodium alginate generally has high viscosity
- a thickener is added to the gelling agent aqueous solution and / or the buffer aqueous solution in advance.
- the thickener dextran, polyethylene glycol, polymethylcellulose, propylene glycol alginate, or any combination thereof can be used.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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. *
- 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.
- 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. )
- 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.
- 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.
- 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. *
- 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. *
- aqueous solution 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. *
- 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.
- aqueous solution 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. *
- aqueous solutions were continuously introduced into the channel structure X using a syringe pump.
- a PTFE tube was used in order to connect each inlet in the syringe and the flow path structure X.
- 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.
- the first aqueous solution introduced from the inlet I1 is 5 to 10 microliters per minute
- the inlet I2 , I2 ′ and I2 ′′ were introduced into the second aqueous solution at 10 to 50 microliters per minute
- 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
- 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.
- 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. *
- the flow rate of the first aqueous solution introduced from the inlet I1 is 3 microliters per minute
- the flow rate of the second aqueous solution introduced from the inlets I2, I2 ′, and I2 ′′ is the total.
- 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.
- 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. *
- 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.
- region C were produced.
- 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.
- 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.
- 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 ⁇ M 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. *
- 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
- the infiltration behavior was significantly different.
- 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.
- 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.
- 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.
- gene expression was quantitatively evaluated by real-time PCR using GAPDH as a housekeeping gene and TaqMan probe as a fluorescent probe.
- GAPDH a housekeeping gene
- TaqMan probe as a fluorescent probe.
- HIF-1a hypooxia-inducing factor
- MMP2 matrix metalloprotease
- VEGF vascular endothelial growth factor
- 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)
- 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.
- 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.
- 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. *
- 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. *
- the present invention 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.
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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
本発明は、細胞評価用ハイドロゲル基材および細胞評価手法に関する。
The present invention relates to a hydrogel substrate for cell evaluation and a cell evaluation method.
癌に対して効果および選択性が高く、また副作用の少ない抗癌剤を開発するためには、膨大な数の新薬候補の中から、有用なものを絞り込むためのスクリーニング操作が必要であり、現在、実験動物を用いた薬効評価を行う前段階として、培養した癌細胞を用いた薬剤アッセイが行われている。
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. *
癌細胞に対し抗癌剤がどの程度作用するかについて評価を行う場合に、癌細胞の浸潤および転移を定量的に評価することが重要である。
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. *
現在、癌細胞の浸潤挙動や転移を解明するためには、マルチウェルプレート等を利用した通常の平面培養のほか、直径数ミクロンの穴の開いたメンブレンを組み込んだマルチウェルプレートを利用した培養手法も利用されており、細胞の生存率あるいは浸潤度に対する、抗癌剤の半数阻害濃度(IC50)の測定などが行われている。
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. *
また、平面における単独培養と比較して、生体内における癌細胞の挙動を、より忠実に生体外において再現するための細胞培養システムに関する研究も盛んに行われている。例として、特許文献1に示されるように、3次元的なハイドロゲルの内部の一定の位置に癌細胞を包埋し、ゲル中における癌細胞の挙動を評価するシステムが提案されており、立体的な環境における浸潤評価や薬効試験が可能となっている。
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. *
また、非特許文献1に示されるように、平面上での培養ではあるものの、癌細胞と他の細胞を共培養するシステムも提案されている。このシステムでは、中央にバルブを有するチャンバー内に、ヒト子宮頸癌細胞およびヒト血管内皮細胞を播種することで、2次元的な環境下において共培養を行っており、複数種の細胞が存在する生体組織に近い環境における癌細胞の挙動の観察が可能となる。
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. *
さらにまた、非特許文献2に示されるように、腫瘍組織内部の低酸素領域が腫瘍の悪性度に関わる様々な遺伝子を発現していることに着目し、癌細胞の球状の凝集体(スフェロイド)を構築し、3次元的な低酸素領域を再現する手法も提案されている。格子状の凹凸構造を底面に有する培養プレート上に様々な種類の癌細胞を播種し、培養することで、均一な大きさの球状の細胞凝集体(スフェロイド)を構築し、生体内の腫瘍内部にしばしば見られる低酸素領域が再現されている。
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.
しかしながら、マルチウェルプレートを用いた通常の薬剤アッセイでは、平面的な培養基材上に播種した癌細胞が用いられているため、3次元的かつ複数種の細胞が高密度で存在する生体内組織の環境を再現できているとは言い難く、抗癌剤の効果を、生理学的環境と同様の条件下で正確に評価することは不可能である。そのため、より生体に近い細胞培養環境において細胞評価を行うシステムや手法の開発が必要とされている。
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. *
また、特許文献1、あるいは非特許文献2に示される技術を用いた場合、3次元的な空間において癌細胞の浸潤評価を行うという意味において、生体環境と一部類似する環境を再現しているものの、癌細胞と正常細胞が相互作用を及ぼしあっている生体内の腫瘍とは大きく異なるため、抗癌剤の薬効を正確に評価することは不可能となる、という問題点がある。
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. *
また、非特許文献1に示される技術を用いた場合、複数種の細胞が存在するという意味において生体内の環境と類似するものの、2次元平面的な細胞培養系であるため、生体内の3次元的な腫瘍組織との相関が得られない、という問題点がある。
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. *
つまり、生体内環境により近い条件下において癌細胞の挙動を評価するためには、これらの技術の利点を兼ね備えた、(1)3次元的な環境下において、(2)複数種の細胞を共培養する、システムを構築することが望ましいが、このような条件を満たすシステムはこれまでに開発されていない。
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. *
さらに、癌細胞の浸潤度合いを評価する上で、癌細胞の初期位置を一定に制御できれば、癌細胞の浸潤を定量的かつ正確に評価する上で非常に有利であるが、マイクロメートルの正確さで、3次元的な培養環境の内部に細胞を配置する手法も、これまでにほとんど開発されていない。
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. *
本発明は、従来の技術の有する上記したような問題点に鑑みてなされたものであり、その目的とするところは、従来技術と比較してより生理的環境に近い条件下において、正確に癌細胞の浸潤挙動を評価するために、3次元的な環境であり、かつ複数種の細胞が共存する新規細胞培養系を構築しようとするものである。
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. *
また本発明は、癌細胞の初期位置を一定の範囲に制限することによって、非常に簡便かつ正確に、癌細胞浸潤の定量評価を行うことを可能とする、新規細胞培養系を提供しようとするものである。
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. *
さらに本発明は、作製した新規細胞培養基材を利用することによって、簡便かつ正確に、生体内の環境と類似した環境において、癌細胞の浸潤評価を可能とする、新規細胞評価手法を提供しようとするものである。
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.
上記目的を達成するための、本発明の一観点に係る発明は、ハイドロゲルによって構成され、ファイバー状、あるいは、シート状の形態を有し、長さ方向の軸に対して垂直な平面における断面Sの内部において、周縁部が断面Sの周縁部に接しておらず、かつ、第一の細胞が存在する、領域Aを有し、さらに、領域Aは、断面Sにおける領域A以外の領域Bと比較して、弾性率の低いハイドロゲルによって構成されている、細胞評価用ハイドロゲル基材である。このような基材を用いることによって、3次元的なファイバー状またはシート状のハイドロゲル基材の内部の特定の位置に、癌細胞などの、評価対象となる第一の細胞を配置することが可能となり、また、第一の細胞が浸潤し増殖する領域および方向を、内部の一定の領域のみに制限することが可能となり、正確な浸潤挙動評価を行うことが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記ハイドロゲル基材は、断面Sにおいて、部分的に領域Aおよび領域Bに接し、かつ、部分的に断面Sの周縁部に接する、領域Cを有し、さらに、領域Cは、領域Bと比較して、弾性率の低いハイドロゲルによって構成されていることが望ましい。このようにすることによって、ハイドロゲル基材中に内包した、評価対象となる第一の細胞の浸潤する方向を、領域Aから領域Cを通じてハイドロゲル基材の外側方向へと制御することが可能となり、より正確かつ簡便に、領域Aに包埋した第一の細胞の浸潤挙動評価を行うことが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、領域Bには、前記第一の細胞とは異なる第二の細胞が存在することが望ましい。このようにすることによって、たとえば第一の細胞として癌細胞の存在する領域Aの周囲に、癌細胞以外の細胞が存在し、癌細胞と相互作用を及ぼしあう環境において、癌細胞がハイドロゲル基材の外部に向かって浸潤する状況を再現することができるため、癌細胞の浸潤挙動を正確かつ簡便に評価することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、領域Cには、前記第二の細胞が存在することが望ましい。このようにすることによって、癌細胞の浸潤方向である領域に、癌細胞以外の細胞が存在する状況を再現することができるため、癌細胞の浸潤挙動を正確かつ簡便に評価することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、領域A、領域B、および領域Cを構成するハイドロゲルは、アルギン酸、あるいは、アルギン酸の誘導体によって構成されていることが好ましい。このようにすることによって、容易に成型しやすく、生体適合性に優れ、細胞の生存を維持しながら細胞を包埋でき、機械的強度が高く、細胞培養液中において比較的長期にわたって安定な、ハイドロゲル基材を利用することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、領域Aおよび領域Cを構成するハイドロゲルには、アルギン酸プロピレングリコールが含まれていることが好ましい。このようにすることによって、弾性率の低い、領域Aおよび領域Cを構成することが可能となり、第一の細胞が領域Aから領域Cを通過してハイドロゲル基材の外へとより効率的に浸潤することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、領域A乃至領域Cを構成するハイドロゲルのマトリックス中には、細胞外マトリックスを構成するタンパク質、あるいは、細胞接着性のペプチド分子の少なくともいずれかが含まれていることが好ましい。このようにすることによって、コラーゲン等のマトリックス成分が含まれる生体環境をより高度に模倣した環境における細胞の浸潤挙動の評価が可能となるほか、細胞のマトリックスへの接着性が向上し、細胞の浸潤が促進され、評価にかかる時間を大幅に短縮することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記第一の細胞とは、癌細胞であることが好ましい。このようにすることによって、癌細胞の浸潤を評価するためのハイドロゲル基材の提供が可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記第二の細胞とは、癌細胞以外の細胞であることが好ましい。このようにすることによって、癌細胞と正常細胞が存在し相互作用を及ぼしあう生体環境と同様の環境における、癌細胞の浸潤挙動の評価が可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記第二の細胞とは、繊維芽細胞、内皮細胞、上皮細胞、平滑筋細胞、骨格筋細胞、中皮細胞、実質細胞、腺細胞、表皮細胞、神経細胞、骨芽細胞、これらの細胞の前駆細胞、各種幹細胞より分化させた細胞、の少なくともいずれかであることが好ましい。このようにすることによって、これらの細胞と癌細胞が相互作用を及ぼしあう生体環境と同様の環境における、癌細胞の浸潤挙動の評価が可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、領域Aの面積は、前記第一の細胞の平均直径をDとすると、10D2以下であることが好ましい。このようにすることによって、領域Aに包埋する第一の細胞の初期位置を、一定の範囲に制限することが可能となるため、その浸潤挙動をより正確かつ簡便に評価することが可能となる。
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.
また、本観点に係る発明において、限定されるわけではないが、領域Aの面積をSとし、領域Aの周囲長をLとすると、領域Aの円形度4πS/L2は0.5以上であることが好ましい。このようにすることによって、領域Aに包埋する第一の細胞の初期位置を、断面における一定の範囲に制限することが可能となるため、その浸潤挙動をより正確かつ簡便に評価することが可能となる。
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.
また、本観点に係る発明において、限定されるわけではないが、前記第一の細胞の平均直径をDとすると、領域Cの幅は3D以下であることが好ましい。このようにすることによって、癌細胞が外部へと浸潤する際に通過する領域Cの幅を制限することができるため、より効率的かつ迅速な癌細胞の浸潤評価が可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、ハイドロゲル基材は、直径500マイクロメートル以下のファイバー状であることが好ましい。こ
のようにすることによって、酸素あるいは栄養分の不足による内部に包埋した細胞の壊死を防ぐことが可能となるほか、癌細胞がハイドロゲル基材の外部へと浸潤するために必要となる時間を短縮することが可能となる。 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.
のようにすることによって、酸素あるいは栄養分の不足による内部に包埋した細胞の壊死を防ぐことが可能となるほか、癌細胞がハイドロゲル基材の外部へと浸潤するために必要となる時間を短縮することが可能となる。 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.
また、本観点に係る発明において、限定されるわけではないが、ハイドロゲル基材は、厚さ500マイクロメートル以下のシート状であり、断面Sの内部において、領域Aは複数個所存在していてもよい。このようにすることによって、ファイバー状のハイドロゲル基材の場合と同様に、細胞の壊死を防ぎ、癌細胞の浸潤時間を短縮することが可能となるほか、第一の細胞が存在する領域Aを複数個所設けることによって、評価対象となる細胞を並列的に配置した、より高密度な細胞評価系の構築も可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、領域Aに含まれる、前記第一の細胞の密度は、1立方センチメートル当たり10万個以上であることが好ましい。このようにすることによって、内部に高密度に癌細胞を包埋したハイドロゲル基材の利用が可能となり、より効率的に癌細胞の浸潤挙動を評価することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、領域B乃至領域Cに含まれる、前記第二の細胞の密度は、1立方センチメートル当たり100万個以上であることが好ましい。このようにすることによって、内部に正常細胞を高密度に包埋したハイドロゲル基材の利用が可能となり、より生体環境に近い状況における癌細胞の浸潤挙動評価が可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、ハイドロゲル基材は、少なくとも2つの入口I1~In(n≧2)と、入口I1~Inにそれぞれ接続する入口流路CI1~CInと、入口流路CI1~CInが同時あるいは段階的に合流する少なくとも1つの合流点P1~Pm(m≧1)と、合流点P1~Pmより下流に存在する合流流路Gと、合流流路Gの下流に存在する出口Oを有する、流路構造Xに対し、アルギン酸ナトリウムを含み、かつ前記第一の細胞を懸濁させた、第一の水溶液を、入口I1を介して、また、アルギン酸ナトリウムを含む第二の水溶液を、入口I2を介して、それぞれ連続的に導入し、流路構造Xの内部において、前記第一の水溶液および前記第二の水溶液を接触させ、さらに流路構造Xの内部乃至外部において、前記第一の水溶液および前記第二の水溶液をゲル化剤水溶液と接触させることによって、前記第一の水溶液および前記第二の水溶液が接触した状態で連続的にゲル化することによって作製されたものであり、また、領域Aは前記第一の水溶液がゲル化することによって形成されたものであり、かつ、領域Bは前記第二の水溶液がゲル化することによって形成されたものであることが望ましい。このようにすることによって、組成の異なる複数の部分によって構成された断面パターンを有し、包埋した細胞の位置が正確に制御され、細胞を生きたまま包埋可能な、ファイバー状あるいはシート状のハイドロゲル基材を、簡便かつ連続的に作製することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、ハイドロゲル基材は、少なくとも3つの入口I1~In(n≧3)と、入口I1~Inにそれぞれ接続する入口流路CI1~CInと、入口流路CI1~CInが同時あるいは段階的に合流する少なくとも1つの合流点P1~Pm(m≧1)と、合流点P1~Pmより下流に存在する合流流路Gと、合流流路Gの下流に存在する出口Oを有する、流路構造Xに対し、アルギン酸ナトリウムを含み、かつ前記第一の細胞を懸濁させた、第一の水溶液を、入口I1を介して、また、アルギン酸ナトリウムを含む第二の水溶液を、入口I2を介して、さらに、アルギン酸ナトリウムを含む第三の水溶液を、入口I3を介して、それぞれ連続的に導入し、流路構造Xの内部において、前記第一の水溶液、前記第二の水溶液、前記第三の水溶液を接触させ、さらに流路構造Xの内部乃至外部において、前記第一の水溶液、前記第二の水溶液、および前記第三の水溶液をゲル化剤水溶液と接触させることによって、前記第一の水溶液および前記第二の水溶液が接触した状態で連続的にゲル化することによって作製されたものであり、また、領域Aは前記第一の水溶液がゲル化することによって形成されたものであり、かつ、領域Bは前記第二の水溶液がゲル化することによって形成されたものであり、さらに、領域Cは前記第三の水溶液がゲル化することによって形成されたものであってもよい。このようにすることによって、断面が組成の異なる少なくとも3つの領域によって構成される、ハイドロゲル基材を利用することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記第二の水溶液乃至前記第三の水溶液には、前記第二の細胞が懸濁されていることが好ましい。このようにすることによって、前記第二の細胞が領域B乃至領域Cに包埋されたハイドロゲル基材を作製することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、流路構造Xは、少なくとも一つの入口G1~Gn(n≧1)および、入口G1~Gnにそれぞれ接続し、合流点P1~Pmのいずれかにおいて合流流路Gに合流する、入口流路CG1~CGnを有しており、入口G1~Gnを介して、前記ゲル化剤水溶液は流路構造Xに連続的に導入されることが好ましい。このようにすることによって、流路構造内部においてゲル化した、より径あるいは厚みの均一なハイドロゲル基材を利用することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、流路構造Xは、少なくとも一つの入口B1~Bn(n≧1)および、入口B1~Bnにそれぞれ接続し、合流点P1~Pmのいずれかにおいて合流流路Gに合流する、入口流路CB1~CBnを有しており、入口B1~Bnを介して、バッファー水溶液は流路構造Xに連続的に導入されることが好ましい。このようにすることによって、より一層径あるいは厚みの均一な、ハイドロゲル基材を利用することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、合流点P1~Pmのうち少なくとも一つの合流点において、前記第一の水溶液は、その上下左右の少なくともいずれかおいて前記第二の水溶液と接触することが好ましい。このようにすることによって、断面が複数の領域によって構成されたハイドロゲル基材をより簡便に形成することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、流路構造Xは、少なくとも部分的にキャピラリー状の管によって構成されていてもよい。このようにすることによって、前記第一の水溶液、前記第二の水溶液、および前記第三の水溶液を、キャピラリー管を通して接触させ、その後ゲル化させることによって、効率的に作製されたハイドロゲル基材を利用することが可能となるほか、任意の位置に並列的に配置されたキャピラリー管や多重管を利用することによって、任意の断面形状を有するハイドロゲル基材を簡便に作製することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、流路構造Xは、少なくとも部分的に、微細加工技術を用いて作製されたマイクロ流路構造によって構成されても良い。このようにすることによって、任意の断面形状を有する、直径あるいは厚さ1ミリメートル以下のハイドロゲル基材を、正確かつ簡便に作製することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、流路構造Xは、その幅、深さ、直径等の値のうち少なくともいずれか一つが、少なくとも部分的に1ミリメートル以下であることが好ましい。このようにすることで、直径100~500マイクロメートル程度のハイドロゲル材料を正確かつ簡便に作製することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記第一の水溶液乃至前記第三の水溶液に含まれるアルギン酸ナトリウムの濃度は、それぞれ100mLあたり1g以下であることが好ましい。このようにすることによって、領域Aおよび領域Cの弾性率が低いハイドロゲル基材を、簡便かつ正確に作製することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記第二の水溶液に含まれるアルギン酸ナトリウムの濃度は、100mLあたり1g以上であることが好ましい。このようにすることによって、領域Bの弾性率が高いハイドロゲル基材を、簡便かつ正確に作製することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記ゲル化剤水溶液は、塩化バリウムを含むことが好ましい。このようにすることによって、ゲル化剤としてカルシウムイオンのみを含むハイドロゲル基材と比較して、リン酸塩等を含む培養液中に浸した際のハイドロゲル基材の膨潤が抑制されるため、包埋した細胞密度を高く保つことが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記ゲル化剤水溶液乃至前記バッファー水溶液は、増粘剤を含むことが好ましい。このようにすることによって、流路内に形成した層流の流れを安定化することが可能となり、径あるいは厚みの均一なハイドロゲル材料の作製および利用が可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記第一の水溶液乃至前記第三の水溶液は、アルギン酸プロピレングリコールを含むことが好ましい。このようにすることによって、断面が弾性率の異なる複数の部分からなるハイドロゲル基材を、簡便かつ正確に作製することが可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記第一の水溶液乃至前記第三の水溶液に含まれるアルギン酸プロピレングリコールの濃度は、それぞれ100mLあたり1g以上であることが好ましい。このようにすることによって、領域Aおよび領域Cの弾性率が十分に低いハイドロゲル基材の作製と利用が可能となる。
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. *
また、本発明の他の一観点に係る発明は、請求項1乃至32のいずれか一項に記載の細胞評価用ハイドロゲル基材を用いる、細胞評価手法である。このような評価法は、複数種の細胞を、3次元的なハイドロゲルのマトリックス中に位置を制御して配置できるため、これまでに利用されている平面的な培養基材を用いた細胞浸潤評価法などと比較して、より生体内の環境に近い条件における細胞の評価が可能となる。
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. *
また、本観点に係る発明において、限定されるわけではないが、前記ハイドロゲル基材内部に包埋した細胞に対し、培養操作を行い、前記第一の細胞が前記ハイドロゲル基材の内部を浸潤しゲル外部にまで達する個数あるいは割合を、計測することによって、細胞の浸潤を定量的に評価することが好ましい。このようにすることによって、ハイドロゲル基材の内部を浸潤し、外部へと増殖した細胞の割合を、簡便、迅速、かつ正確に定量することが可能となるため、癌細胞等の細胞の浸潤挙動を評価する上で、非常に効果的である。
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.
本発明は、以上に述べられたように構成されているため、癌細胞の浸潤評価を行う上で有用な、癌細胞が断面内部の任意の位置に配置され、その周囲が他の細胞によって取り囲まれた、ファイバー状あるいはシート状のハイドロゲル基材の利用が可能となる。そして、ハイドロゲル基材中の細胞に対し培養操作を行うことによって、既存の細胞培養手法では不可能であった、3次元的に密接した状態における癌細胞と癌細胞以外の細胞の培養操作が可能となる。
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. *
また本発明は、以上に述べられたように構成されているため、ハイドロゲル基材中に包埋した癌細胞の初期位置を一定の範囲内に制御することが可能となるほか、ハイドロゲル基材の断面に設けられた弾性率の低い軟化部分や、内部に導入された細胞外マトリックス成分を利用することによって、癌細胞の浸潤を促進することが可能と
なる。そのため、既存の手法やシステムと比較して、癌細胞の浸潤挙動をより再現性高く、正確に、簡便に、かつ効率的に評価することが可能となる。 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.
なる。そのため、既存の手法やシステムと比較して、癌細胞の浸潤挙動をより再現性高く、正確に、簡便に、かつ効率的に評価することが可能となる。 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.
さらに本発明は、以上に述べられたように構成されているため、微細な流路構造を利用して正確かつ簡便に作製されたシート状あるいはファイバー状のハイドロゲル基材を利用することが可能となる。そのため、ハイドロゲル基材を作製する際に、包埋される細胞の位置を制御するための複雑な装置や機構を必要としない、という優れた効果を発揮する。
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. *
さらに本発明は、以上に述べられたように構成されているため、包埋される細胞の位置が正確に制御されたハイドロゲル基材を利用した、生体内により近い環境における細胞の浸潤挙動を評価するための、正確かつ簡便な手法の提供が可能となる。そのため、たとえば培養時に、必要に応じて抗癌剤等の薬剤を培養液に添加することで、生体内環境に近い状態における薬剤アッセイを行うことが可能となるほか、癌細胞研究のためのモデルとしての利用が可能となり、創薬分野において、あるいは細胞生理学の研究において有用な、新規手法を提供できる。
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.
以下、本発明に係る細胞評価用ハイドロゲル基材および細胞評価方法の最良の形態を詳細に説明するものとする。ただし、本発明は多くの異なる形態による実施が可能であり、以下に示す実施形態、実施例の例示にのみ限定されるものではない。
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. *
図1には、ファイバー状の細胞評価用ハイドロゲル基材の一例を示した概略図が示されており、図1(a)はファイバー状のハイドロゲルの一部分を立体的に示した概略図であり、図1(b)は細胞評価用ハイドロゲル基材の図1(a)における平面bにおける断面Sを示した概略図であり、図1(c)は細胞評価用ハイドロゲル基材の平面cにおける断面図である。なお、本図において、内部に包埋した細胞は描かれていない。
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. *
図1に示すハイドロゲル基材は、弾性率が相対的に低いハイドロゲルによって構成された領域Aおよび領域Cと、弾性率が相対的に高いハイドロゲルによって構成された領域Bによって構成されている。
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. . *
図1に示すハイドロゲル基材は、アルギン酸を主成分とする高分子によって形成されている。アルギン酸は、たとえばカルシウムやバリウムといった、多価のカチオンの存在下において、迅速にゲル化し、また、ゲル化時、ゾル化時、あるいは溶解時に、温度制御を行う必要がないため、細胞の生存および機能を維持しながら細胞をハイドロゲル基材中に包埋する上で、都合が良い。なお、アルギン酸以外にも、天然あるいは合成の高分子を利用して形成されたハイドロゲル基材を用いることも可能であり、たとえば、ポリエチレングリコールジアクリレートの重合によって形成された高分子や、ポリアクリルアミド、アガロース、コラーゲン、架橋ゼラチンなどを用いることも可能である。さらに、これらの高分子のうち複数種を用いて形成されたハイドロゲル基材を用いることも可能である。
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. *
なお、アルギン酸を用いて形成されたハイドロゲル基材の場合には、領域Aおよび領域Cを構成するハイドロゲル基材中に、アルギン酸プロピレングリコールが含まれていることが好ましい。アルギン酸プロピレングリコールは、アルギン酸のエステル誘導体であり、多価のカチオンの存在下でもゲル化しにくいという特性をもつ。そのため、アルギン酸プロピレングリコールを、アルギン酸に対し一定量添加することによって、ハイドロゲル基材の作製時に、ゲル化時に生じるアルギン酸の収縮防ぎ、かつ、領域Aおよび領域Cの弾性率を効率的に低下させることが可能である。
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. *
図2には、2種類の細胞を包埋した細胞評価用ハイドロゲル基材における、培養操作前後の細胞の様子を模式的に示した概略図が示されており、図2(a)は、培養前後における、ハイドロゲル基材の断面を立体的に示した模式図であり、図2(b)は、培養前後における、ハイドロゲル基材の、長さ方向の軸と直行する平面における断面を示した模式図である。
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. *
図2に示すように、ハイドロゲルの中心部付近に位置する領域Aに包埋した、評価対象となる第一の細胞は、培養操作を行うことによって、ハイドロゲル基材の外側方向へと移動および/あるいは増殖し、浸潤する。なおこの際、ハイドロゲル基材における領域Aは、ハイドロゲル基材の断面Sの周縁部に接触していなければ良いため、中心部付近に位置していなくても構わない。
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. *
領域Aの内部に包埋する第一の細胞として、癌細胞を用いることで、癌細胞の浸潤評価を行うことが可能であり、たとえば、新規抗癌剤の探索のためのスクリーニングや薬効評価、あるいは癌細胞の生理学的解析を行うことが可能となる。癌細胞としては、各種培養細胞株を用いることができるほか、必要に応じてプライマリの細胞を用いることも可能である。また、癌細胞に限らず、各種幹細胞や増殖能の高い正常細胞を用いることで、それらの評価を行うことも可能である。
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. *
なお、ハイドロゲル基材における領域Bおよび領域Cに、第二の細胞を包埋せずとも、第一の細胞を単独で培養することで、3次元的な環境におけるその浸潤挙動の評価を行うことが可能である。しかしながら、領域Bおよび領域Cに第二の細胞を包埋することによって、より生体の組織を模倣した環境における癌細胞の浸潤評価を行うことが可能となる。
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. *
第二の細胞としては、各種培養細胞やプライマリ細胞などの、任意の細胞を用いることが可能である。しかし、対象となる癌細胞に応じて、その種類を適切に選択することが好ましい。第二の細胞の例としては、繊維芽細胞、内皮細胞、上皮細胞、平滑筋細胞、骨格筋細胞、中皮細胞、実質細胞、腺細胞、表皮細胞、神経細胞、骨芽細胞、これらの細胞の前駆細胞、各種幹細胞より分化させた細胞等を用いることが可能である。
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. *
また、ハイドロゲル基材を構成するマトリックス中に、コラーゲン、フィブロネクチン、ラミニン等の細胞外マトリックス分子や、マトリゲル等の細胞外マトリックス混合物などを添加することが可能であり、また、ハイドロゲルを構成する主成分として、細胞接着性のRGD等のペプチドが共有結合したアルギン酸等を用いることもできる。このようにすることで、細胞接着性のマトリックスが豊富に存在する生体内の環境をより高度に模倣できるほか、特に領域AおよびCにこれらの成分を導入することによって、癌細胞のマトリックスへの接着を高め、浸潤速度を増加し、さらに、浸潤に必要な時間を短縮することが可能となる。
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. *
ハイドロゲル基材の全体の大きさは、癌細胞の浸潤評価を行うことができる範囲であれば、どの程度であっても構わない。しかし、外部に出てきた細胞の割合を計測することによってその浸潤度合いを評価する場合には、ハイドロゲル基材の直径あるいは厚みは、値がある程度小さい方が好ましい。ただし、酸素欠乏の度合いを測定する際には、領域Bに包埋された第二の細胞によって、中心部に位置する癌細胞に対し酸素の供給が不足する欠乏する程度の値である
ことが好ましい場合もありうる。これらの観点から、ハイドロゲルの直径は、50マイクロメートル以上500マイクロメートル以下であることが好ましく、50マイクロメートル以上300マイクロメートル以下であることがより好ましい。 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.
ことが好ましい場合もありうる。これらの観点から、ハイドロゲルの直径は、50マイクロメートル以上500マイクロメートル以下であることが好ましく、50マイクロメートル以上300マイクロメートル以下であることがより好ましい。 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.
なお、ハイドロゲル基材は、その長さ方向において、部分的に径あるいは厚みが不均一であっても良く、また、断面における領域A、領域B、および領域に変化が生じていても良く、また、途中に分岐を有するような形態であっても良く、さらには、各領域の化学的組成が異なっていても良い。しかし、断面およびそれを構成する領域の形状および組成が均一なハイドロゲル基材は、包埋した細胞の培養時の諸条件を均一にできるという観点から、より好ましい。
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. *
ハイドロゲル基材の長さは、用途に応じて、適切な長さとすることが好ましい。ただし、信頼性の高い評価実験を行うためには、評価対象となる第一の細胞が、100個以上存在することが好ましく、300個以上存在することがより好ましい。このような観点から、ハイドロゲル基材の長さは、1ミリメートル以上であることが好ましく、10ミリメートル以上であることがより好ましい。
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. *
断面Sにおける領域Aの面積は、包埋した第一の細胞の初期位置を一定に制限するという観点から、前記第一の細胞の平均直径をDとすると、10D2以下であることが好ましい。
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.
また、領域A内に包埋した第一の細胞が存在する位置の違いによって、培養条件の違いが生じさせることを防ぐという観点から、断面Sにおける領域Aの相対的な位置は、断面Sの周縁部から比較的均一な距離にあることが望ましい。そのため、領域Aの面積をSとし、領域Aの周囲長をLとすると、領域Aの円形度4πS/L2は0.5以上であることが好ましい。
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.
また、領域Cの幅が細胞の平均直径と近い値となることによって、細胞の浸潤方向が一定方向に制御される効率が高まるため、前記第一の細胞の平均直径をDとすると、領域Cの幅は3D以下であることが好ましい。
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. *
領域Aに包埋する第一の細胞の密度は、その値があまりに低すぎる場合には、細胞の評価を行う効率が下がる。そのため、その密度は高いほうが好ましく、ハイドロゲル基材1立方センチメートル当たり10万個以上であることが好ましい。ただし、密度があまりに高すぎると、長さ方向において隣り合う細胞同士が互いに接着して一つのコロニーを形成してしまうため、一定の値以下であることも必要となる。この場合の一定の値とは、たとえば、ハイドロゲル基材1立方センチメートル当たり1000万個であり、あるいは、隣り合う細胞間の平均距離が、100マイクロメートル以下となるような密度である。
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. *
領域B乃至領域Cに包埋する第二の細胞の密度は、細胞が高密度にパッキングされた生体組織を模倣するという観点から、高いほうが好ましく、ハイドロゲル基材1立方センチメートル当たり100万個以上であることが好ましい。第二の細胞に関しては、長さ方向において隣り合う細胞同士が互いに接着して一つのコロニーを形成しても構わない。
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. *
図2に示すように、適切な培養操作を行うことによって、癌細胞が外部へと浸潤する。その後、必要に応じて3次元的な培養環境における癌細胞の浸潤度合いを定量的に評価することで、細胞の評価を行うことが可能となる。浸潤度合いの定量化のための最も簡便な手段は、顕微鏡などを用いて、外部へと浸潤した細胞を観察し、その割合を単純に計測することである。また、それ以外にも、染色などによる生存率の評価、画像解析技術を利用したハイドロゲルマトリックス中の浸潤度合いの定量、リアルタイムPCRによる遺伝子発現の定量、などを、独立に、あるいは同時に行うことも可能である。
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. *
細胞の培養操作としては、作製したハイドロゲル基材を培養液中に浸して、制御された温度、酸素濃度、炭酸ガス濃度条件下において保持することが好ましい。培養操作において用いる培養液としては、対象とする細胞種に応じた一般的な細胞培養用培養液を用いることができる。また、培養時には、ハイドロゲル基材を、シャーレやフラスコ等に満たした培養液中に浸し、CO2インキュベーター等の培養装置を用いて培養することが好ましい。さらに、必要に応じて振盪操作を行うことも可能である。
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. *
また、たとえば薬剤が細胞の浸潤に与える影響を評価する際には、薬剤を培養液中に添加して培養を行うことで、培養液中の薬剤濃度と浸潤度合いの定量的評価を、簡便に行うことが可能である。また、ハイドロゲル基材を一定時間、薬剤を含む溶液中に浸漬した後に、薬剤を含まない培養液中で培養することによって、一定の時間、薬剤を作用させ、その影響を評価することも可能である。
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. *
なお、ハイドロゲル基材としては、図1および図2に概略図を例示したもの以外にも、様々な形状のものを用いることが可能である。
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. *
図3には、ファイバー状あるいはシート状のハイドロゲル基材例を示した概略図が示されており、図3(a)は、断面が円形であり、かつ領域Aおよび領域Bからなるハイドロゲルファイバーの概略図であり、図3(b)は、断面が矩形であり、かつ領域A、領域B、および領域Cからなるハイドロゲルファイバーの概略図であり、図3(c)は、一定の厚みを有し、複数の領域Aおよび領域C、および領域Bからなる、ハイドロゲシートの概略図である。
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. *
図3(a)のようなハイドロゲル基材を用いることで、領域Aに包埋した第一の細胞が、ハイドロゲル基材の外側ではなく、領域A内部を、ハイドロゲル基材の長さ方向に浸潤する様子を観察することが可能となる。また、図1に示したような、領域A、領域B、領域Cの3つの領域からなるハイドロゲル基材の場合、図3(b)に示すように、断面が矩形であっても良く、さらに、図3(c)に示すように、シート状のハイドロゲル基材であって、その内部に、第一の細胞を含む領域Aを複数有するものであっても良い。特に、図3(c)に示すシート状のハイドロゲル基材の場合は、平面的な形態の中に、評価対象となる第一の細胞を高密度かつ高効率に包埋することが可能となり、細胞の評価をより効率的に行うことが可能となる。
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. *
これらのハイドロゲル基材を作製するプロセスとして、マイクロ流路構造を用いるものは非常に効率的である。
As a process for producing these hydrogel substrates, those using a microchannel structure are very efficient. *
図4には、流路構造を利用した、細胞評価用ハイドロゲル基材を作製する様子を示した模式図が示されており、図4(a)は、平面的に形成された流路構造Xを状面から観察した様子、および、その内部における溶液および細胞の流れの様子を模式的に示した概略図であり、図4(b)は、図4(a)におけるA-A’線における流路構造Xの断面を示した模式図であり、図4(c)は、図4(a)におけるB-B’線における流路構造Xの断面を示した模式図である。
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. *
図4に示す流路構造Xは、8つの入口I1、I2、I2’、I3、B1、B1’、G1、G1’にそれぞれ接続される入口流路CI1、CI2、CI2’、CI3、CB1、CB1’、CG1、CG1’と、入口流路が段階的に合流する合流点P1、P2、P3、P4と、合流点P4と出口Oの間に存在する合流流路G、を有している。
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. . *
なお、図4に示すような平面的に構成された流路構造を作製する場合、デバイスの材質としては、PDMS(ポリジメチルシロキサン)、アクリル等の各種ポリマー材料、ガラス、シリコン、セラミクス、ステンレスなどの各種金属、などを用いることができ、また、これらの材料のうちの任意の複数種類の基板を組み合わせて用いることも可能である。ただし、平面的な流路を安価に作製するためには、少なくとも部分的にポリマー材料を用いることが好ましい。また、流路構造の加工技術としては、モールディングやエンボッシングといった鋳型を利用した作製技術は、流路構造を容易に作製可能であるという点において好ましいが、その他にも、ウェットエッチング、ドライエッチング、レーザー加工、電子線直接描画、機械加工等の作製技術を用いることも可能である。
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. *
図4に示した流路構造は、作製対象とするハイドロゲル基材の大きさに応じて、適切な内径、幅、深さとなるように設計する必要がある。ただし、直径あるいは厚みが500マイクロメートル以下のハイドロゲル基材を効率的に作製するためには、流路構造の径、深さ、あるいは幅等のいずれかの値は、1ミリメートル以下であることが好ましく、500マイクロメートル以下であることがより好ましい。
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. *
図4に示した流路構造Xに対し、ハイドロゲルを構成するためのゾル水溶液であり、かつ細胞を懸濁させた、第一の水溶液、第二の水溶液、第三の水溶液を、それぞれ入口I1、入口I2およびI2’、入口I3を通して連続的に導入する。また、ゲル化剤水溶液を入口G1および入口G1’から、バッファー水溶液を入口B1および入口B1’から、それぞれ連続的に導入する。
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. *
合流点P4おいて、導入流量を適切に調節することによって、第一の水溶液、第二の水溶液、および第三の水溶液は、それぞれが接した状態において層流を形成する。そして、その下流における合流流路Gにおいて、外側からゲル化剤が拡散してくることによって、これらの水溶液は次第にゲル化し、出口Oより、連続的にファイバー状のハイドロゲル基材が形成される。
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. . *
入口流路構造の立体的配置、分岐点P1およびP2における流路の3次元的構造、および各導入流量等を工夫することによって、流路途中において、第一の水溶液、第二の水溶液、第三の水溶液が、任意の配置となるようにすることが可能であり、その配置を保ったままこれらの溶液をゲル化させることによって、そのパターンを保持したハイドロゲル基材を得ることが可能である。たとえば、合流流路Gにおいて、図4(b)に示すような断面パターンを形成することによって、図1および図2に示すようなハイドロゲル基材を作製することが可能となる。
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. *
ゲル化剤水溶液に含まれるゲル化剤としては、アルギン酸をゲル化することのできる多価の金属カチオンであれば、任意のものを用いることが可能である。しかしながら、細胞毒性の観点から、それらのイオンはカルシウム、ストロンチウム、バリウム、マグネシウムうちのいずれかの2価カチオン、あるいはそれらの任意の混合物であることが好ましい。また、これらのイオンは水溶性である必要があるため、ゲル化剤水溶液は、それらの塩化物を溶解した水溶液であることが好ましい。中でも、作製したハイドロゲル材料の培養液中での膨潤を抑制し、ハイドロゲル基材中の細胞密度を高く保つ、という観点から、ゲル化剤水溶液は塩化バリウムを含むことがより好ましい。なお、細胞へのダメージを低減するためにも、ゲル化剤水溶液の浸透圧はあらかじめ最適な値に調節されていることが望ましい。
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. *
なお、図4に示した流路構造Xは、その内部においてゲル化を行うものであるため、流路途中から、ゲル化剤水溶液を導入できるように設計されており、また、アルギン酸の急速なゲル化による流路の閉塞を防ぐために、ゲル化剤を含まないバッファー水溶液を導入できるように設計されている。しかしながら、これらの水溶液を導入せず、入口B1、B2’、G1、G1’を閉鎖し、出口Oをゲル化剤溶液に浸しておくことで、流路外においてゲルを形成しても良い。ただし、得られるファイバー状のハイドロゲル基材の径を正確に調節しやすい、という観点から、このように流路内部においてゲル化を行う系は好ましい。
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. *
また、アルギン酸ナトリウムを含む水溶液は一般的に高粘度であるため、ゲル化剤水溶液および/あるいはバッファー水溶液には、予め増粘剤が添加されていることが好ましい。なお、増粘剤としては、デキストラン、ポリエチレングリコール、ポリメチルセルロース、アルギン酸プロピレングリコール、あるいはそれらのうちの任意の組み合わせを用いることが可能である。これらの増粘剤をゲル化剤水溶液やバッファー水溶液に添加すること
によって、図4に示すように流路内でゲル化を行う際に、層流を安定的に形成することが可能となり、ハイドロゲル材料の作製時の操作性を向上させることが可能である。なお、第一の水溶液、第二の水溶液、第三の水溶液、ゲル化剤水溶液、バッファー水溶液のうち、室温において、粘度の最小のものと最大のものの粘度の比が、1:1~1:100の範囲にあることが好ましく、1:1~1:10の範囲にあることがより好ましい。なお、流路外においてゲル化を行う場合には、この限りではない。 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.
によって、図4に示すように流路内でゲル化を行う際に、層流を安定的に形成することが可能となり、ハイドロゲル材料の作製時の操作性を向上させることが可能である。なお、第一の水溶液、第二の水溶液、第三の水溶液、ゲル化剤水溶液、バッファー水溶液のうち、室温において、粘度の最小のものと最大のものの粘度の比が、1:1~1:100の範囲にあることが好ましく、1:1~1:10の範囲にあることがより好ましい。なお、流路外においてゲル化を行う場合には、この限りではない。 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.
なお、図4に示した平面的な流路構造以外にも、キャピラリー管を並列化あるいは多重化させたような流路構造を用いることも可能である。
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. *
図5には、シート状の細胞評価用ハイドロゲル基材を作製するための、並列に配置されたキャピラリー管によって一部が構成された流路構造Xにおける、キャピラリー管の部分を模式的に示した概略図が示されており、図中の数字1、2、3は、それぞれ、第一の水溶液、第二の水溶液、第三の水溶液を吐出する出口である。このような並列化キャピラリー構造を用いて、第一の水溶液、第二の水溶液、第三の水溶液を合流させ、その後連続的にゲル化剤水溶液中へと吐出することによって、図3(d)に示すような、シート状のハイドロゲル構造を作製することも可能である。
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. *
なお、以上のような流路構造に導入する、第一の水溶液、第二の水溶液、および第三の水溶液に含まれるアルギン酸は、ナトリウム塩のものを用いることが一般的である。アルギン酸としては、多価のカチオンの存在下でハイドロゲルを形成するものであれば、どのような分子量のものを用いても良い。しかしながら、ハイドロゲル作製時の操作性の観点から、水100mLにアルギン酸1gを溶解し室温に保った場合の粘度が、10cP~400cPの範囲にあるものが好ましい。また、作製されたゲルの強度が一定以上であれば、作製したハイドロゲル材料を培養液中に浸した際の膨潤の程度が低くなり、細胞を高密度で包埋する上で都合が良いため、アルギン酸ポリマーを構成する単位であるウロン酸のうち、グルロン酸の割合が60%以上のものを用いることが好ましい。
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. *
第一の水溶液および第三の水溶液に含まれるアルギン酸ナトリウムの濃度は、領域Aおよび領域Cを形成するハイドロゲルの弾性率を低く保つためにも、各溶液100mLあたり、1g以下であることが好ましく、さらには0.5g以下であることがより好ましい。また、これらの水溶液に含まれるアルギン酸プロピレングリコールの濃度は、領域Aおよび領域Cを形成するハイドロゲルの弾性率を低くするためにも、各溶液100mLあたり、1g以上であることが好ましい。
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. *
一方で、第二の水溶液に含まれるアルギン酸ナトリウムの濃度は、領域Bを形成するハイドロゲルの弾性率を高めるためにも、溶液100mLあたり、1g以上であることが好ましい。また、第二の水溶液は、アルギン酸プロピレングリコールを含まないことが好ましい。
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.
以下、上記実施形態に係る細胞評価用ハイドロゲル基材を作製し、細胞評価を実際に行うことで、本発明の効果を確認した。以下説明する。
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. *
図6には、ファイバー状の細胞評価用ハイドロゲル基材を作製するために利用した、4枚のアクリル板を重ね合わせて作製された流路構造Xを有するマイクロ流体デバイスを示した概略図が示されており、図6(a)~図6(d)は、それぞれ、上から一層目のアクリル板の下面に形成された流路構造、二層目のアクリル板の下面に形成された流路構造、三層目のアクリル板の上面に形成された流路構造、三層目の流路構造の下面に形成された流路構造を、それぞれ示した概略図であるとともに、図6(f)および図6(g)におけるマイクロ流体デバイスのC矢視図であり、図6(e)は、図6(d)における領域eの拡大図であるとともに、図6(f)および図6(g)におけるマイクロ流体デバイスのC矢視図であり、図6(f)および図6(g)は、それぞれ、マイクロ流体デバイスの、図6(a)~図6(e)における、A-A’線、B-B’線における断面図である。
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. *
図6に示した流路構造は、微細加工技術を用いて切削を施した、厚さ2ミリメートルのアクリルの平板を3枚と、切削を施していない厚さ2ミリメートルのアクリルの平板を、熱圧着により積層化することによって形成されている。
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. *
流路構造Xの各部分における流路幅は、たとえば、一層目の平板の下面に存在する流路の幅及び深さは、100マイクロメートル~500マイクロメートルであった。二層目の平板の下面に存在する流路は、幅5ミリメートル、3.5ミリメートル、1.5ミリメートルのものがあり、深さはそれぞれ900マイクロメートル、1.2ミリメートル、1.5ミリメートルであった。三層目の平板の上面に存在する流路の幅及び深さは、500マイクロメートルであった。三層目の平板の下面に存在する流路の幅及び深さは、100マイクロメートル~500マイクロメートルであった。なお、これらの値のうち、一層目の平板の下面に存在する流路の径によって、得られるファイバー状のハイドロゲル基材の径が制御されるため、必要に応じてより太い、あるいはより細い流路構造を使用することが好ましい。
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. *
癌細胞としては、ヒト肺胞基底上皮腺癌細胞であるA549を用い、癌細胞以外の細胞としては、チャイニーズハムスター由来肺繊維芽細胞株である、V79-379A細胞を用いた。これらの細胞は、予め通常の細胞培養ディッシュ上で培養することで、一定量増殖させ、酵素処理によってプレートから剥離し、遠心分離によって培養液を除くことによって調製した。
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. *
第一の水溶液および第三の水溶液としては、水50mLに対し、アルギン酸プロピレングリコールを0.5g、RGDアルギン酸を0.1g、塩化ナトリウムを0.45g、HEPESを5mM、それぞれ溶解させた水溶液を用い、A549細胞を1mLあたり約3000万個、V79-379A細胞を1mLあたり約2億個、それぞれ懸濁させた。なお、これらの成分のうち、RGDアルギン酸は、細胞機能の維持および浸潤の促進のため、HEPESはpH調整のため、また塩化ナトリウムは浸透圧調整のため、それぞれ使用したが、必要に応じて他の成分を加える、あるいはこれらを他の成分と置き換えることも可能である。
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. *
第二の水溶液としては、水50mLに対し、アルギン酸ナトリウムを0.5g、RGDアルギン酸を0.1g、塩化ナトリウムを0.45g、HEPESを5mM、それぞれ溶解させた水溶液を用い、V79-379A細胞を、1mLあたり約2億個懸濁させた。
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. *
また、ゲル化剤水溶液としては、ゲル化剤として塩化バリウムを20mM、増粘剤として分子量50万のデキストランを10%(w/v)、浸透圧調整のための塩化ナトリウムを120mM、pH調整のためのHEPESを10mM含む水溶液を用いた。
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. *
バッファー水溶液としては、増粘剤として分子量50万のデキストランを10%(w/v)、浸透圧調整のための塩化ナトリウムを150mM、pH調整のためのHEPESを10mM含む水溶液を用いた。
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. *
なおこれらの溶液すべては、予め加熱あるいはフィルター処理を施すことによって滅菌操作を行ったものである。
All of these solutions were sterilized by heating or filtering in advance. *
これらの水溶液を、シリンジポンプを用いて、流路構造Xに連続的に導入した。なお、シリンジと、流路構造Xにおける各入口を接続するために、PTFEチューブを使用した。
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. *
各入口から導入した水溶液の流量は、作製対象とする細胞評価用ハイドロゲル基材のサイズに応じて変化させた。各入口からの導入流量は、合流流路Gの幅が300マイクロメートル、深さが300マイクロメートルの場合には、入口I1より導入した第一の水溶液は5~10マイクロリットル毎分、入口I2、I2’、およびI2’’より導入した第二の水溶液は各10~50マイクロリットル毎分、入口I3より導入した第三の水溶液は5~20マイクロリットル毎分、入口G1より導入したゲル化剤水溶液は50~300マイクロリットル毎分、入口B1より導入したバッファー水溶液は5~20マイクロリットル毎分であった。
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. *
入口I1から導入された第一の水溶液は、入口流路CI1を通過して、三層目の平板の下面に形成された垂直上方向の流路構造へと導入され、三層目の平板の上面に存在する、合流点P1において、入口I3から導入され入口流路CI3を通過した第3の水溶液と合流した。さらに、入口I2、I2’、およびI2’’から導入された第二の水溶液と合流点P2において合流し、一層目の平板の下面に存在する合流点P3においてバッファー水溶液と、さらに下流の合流点P4においてゲル化剤水溶液と、それぞれ合流した。そして、合流流路Gの内部において、第一の水溶液、第二の水溶液、第三の水溶液が合流した流れはゲル化し、連続的にファイバー状のハイドロゲル基材が形成され、出口Oより回収された。
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. *
図7には、図6に示された流路構造を用いて作製した、断面が領域A、B、Cからなるファイバー状のハイドロゲル基材中の、細胞培養前後における様子を示した顕微鏡写真が示されており、図7(a)は、領域Aに癌細胞を包埋したハイドロゲル基材の、作製直後の顕微鏡写真であり、図7(b)は、図7(a)に示したハイドロゲル基材の、細胞培養1週間後の顕微鏡写真であり、図7(c)は、領域Aに癌細胞を包埋し、領域Bおよび領域Cに繊維芽細胞を包埋したハイドロゲル基材の、作製直後の顕微鏡写真であり、図7(d)は、図7(c)に示したハイドロゲル基材の、細胞培養1週間後の顕微鏡写真である。
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. *
図7に示したハイドロゲル基材は、入口I1より導入した第一の水溶液の流量が3マイクロリットル毎分、入口I2、I2’、およびI2’’より導入した第二の水溶液の流量が合計72マイクロリットル毎分、入口I3より導入した第三の水溶液の流量が15マイクロリットル毎分、入口B1より導入したバッファー水溶液の流量が25マイクロリットル毎分、入口G1より導入したゲル化剤水溶液の流量が100マイクロリットル毎分の場合に得られたものである。図7(a)に示すように、平均直径約120マイクロメートルのハイドロゲル基材が得られ、その中心部である領域Aに、癌細胞であるA549細胞が長さ方向に間隔をあけて配置されることが観察された。また、第二の水溶液および第三の水溶液に肺繊維芽細胞であるV79-379A細胞を懸濁させて作製したハイドロゲル基材の場合には、A549細胞の周囲にV79-379A細胞が密に配置されている様子が確認された。
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. *
また、図7に示すように、CO2濃度5%雰囲気下において培養液を用いて1週間培養を行ったところ、肺繊維芽細胞を包埋した場合、および、包埋しない場合のいずれの条件でも、中心の癌細胞が領域Cに相当する弾性率の低いハイドロゲル部分を外部へと浸潤し、図2に示すようにハイドロゲル基材の外部にコロニーを形成する様子が確認された。
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.
ハイドロゲル内の癌細胞が、繊維芽細胞が包埋されたゲル内を浸潤し、ゲル外部に飛び出した数を、顕微鏡画像を用いて計測することで癌細胞浸潤挙動を評価したところ、正確かつ簡便に、癌細胞の浸潤割合を評価することが可能であった。
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. *
また、断面における領域Aに癌細胞を包埋したハイドロゲル基材、また、領域Aに癌細胞を、領域Bおよび領域Cに繊維芽細胞を包埋したハイドロゲル基材を作製した。細胞を5日間培養した後、抗癌剤としてシスプラチン培地に添加して24時間培養した後に、さらにシスプラチンを含まない培地を用いて細胞を24時間培養した。その後、抗癌剤が癌細胞の浸潤挙動に与える影響を定量的に評価するため、包埋した癌細胞の数に対する、ファイバー外部に浸潤した癌細胞の割合を、総計300個の癌細胞についてカウントし、浸潤割合を算出した。また、領域Bおよび領域Cに繊維芽細胞を包埋せずに単独培養を行い、共培養との比較を行った。
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. *
図8は、シスプラチン濃度を0.1~100μMの間で変化させた際の癌細胞の浸潤割合について、図6に示される流路構造を用いて作製したハイドロゲル基材内部において繊維芽細胞と共培養した場合および共培養せずに癌細胞を単独で培養した際のそれぞれの条件におけるデータを示したグラフであり、縦軸の浸潤割合比とは、抗癌剤を投与せずに培養した条件においてファイバー外部へと浸潤した細胞数に対する、各抗癌剤濃度条件においてファイバー外部へと浸潤した細胞数の相対的な割合を示している。また、各データは独立した4つのサンプルについてそれぞれ300個の細胞をカウントして得られた値について、その平均値±標準偏差を示している。
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. *
図8に示すように、抗癌剤の濃度に応じて癌細胞の浸潤度合いが異なること、および、ハイドロゲル基材内部において癌細胞を単独培養した場合と、癌細胞および繊維芽細胞を共培養した場合を比較すると浸潤挙動が大きく異なること、などが観察された。特に、2種類の細胞を共培養した系では、抗癌剤濃度が10-5M以上の場合に浸潤割合比が大きく低下した、つまり抗癌剤が効果的に作用したのに対し、癌細胞を単独培養した系では、当該濃度は10-6M以上の場合であり、大きな差が観察された。これらの結果から、共培養と単独培養では、抗癌剤の薬効が大きくことなることが観察された。
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.
また、癌細胞の機能を評価するために、ハイドロゲル基材内部において繊維芽細胞と共培養した癌細胞、および、通常の細胞培養プレートを用いて平面培養した癌細胞を準備し、上記実験と同様の条件で抗癌剤としてシスプラチンを投与した。さらに、mRNAを抽出し、cDNAを合成した後に、それぞれハウスキーピング遺伝子としてGAPDH、蛍光プローブとしてTaqManプローブを用いて、リアルタイムPCRによって遺伝子発現の定量評価を行った。なお、対象として、HIF-1a(低酸素誘導因子)、MMP2(マトリックスメタロプロテアーゼ)、およびVEGF(血管内皮細胞増殖因子)の3種類の遺伝子について定量評価を行った。
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.
図9には、図6に示される流路構造を用いて作製した、断面が領域A、B、Cからなるファイバー状ハイドロゲル基材内部において繊維芽細胞と共培養した癌細胞、および、平面培養した癌細胞に対し、シスプラチンを投与した際の、癌細胞特異的な遺伝子発現を定量評価した結果を示したグラフが示されており、図9(a)はHIF-1a(低酸素誘導因子)、図9(b)はMMP2(マトリックスメタロプロテアーゼ)、図9(c)はVEGF(血管内皮細胞増殖因子)の遺伝子発現をそれぞれ定量した結果を示したグラフである。なお、縦軸の相対的遺伝子発現量とは、平面培養においてシスプラチンを投与しなかった場合の各遺伝子発現量を基準として示した相対値である。
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.
図9に示すように、平面培養した癌細胞では、シスプラチン濃度が100μMである条件において、全ての遺伝子発現量が大きく減少したことが確認された。一方で、ハイドロゲル基材内部において繊維芽細胞と共培養した癌細胞では、抗癌剤を100μM投与した際においても発現量は大幅に低下することなく、これらの遺伝子発現が活発であることが確認された。これは、3次元的な環境かつ、癌細胞周囲に正常細胞が存在する、生体内に近い環境を模倣しているためであると考えられ、この結果からも、本発明において提案した3次元的な共細胞培養環境の優位性および生体との類似性が実証された。
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.
本発明は、以上説明したように構成されているため、創薬のスクリーニングや、細胞を用いた薬剤代謝・毒性評価試験などにおいて通常用いられる、平面的な培養基材を利用した細胞培養系と比較して、3次元的かつ生体を模倣した環境における細胞の浸潤挙動の評価を簡便かつ正確に行うことが可能となる。通常の平面培養は、操作や装置がシンプルになるという利点はあるものの、その環境は、細胞にとって通常の生体環境とは大きく異なるため、細胞を用いたアッセイを行う上で生体内の環境を正確に再現しているとは言い難い。そのため、本発明による3次元的なハイドロゲル基材は、生体構造を反映した環境において細胞アッセイを簡便かつ正確に行うためのツールとして、製薬産業のみならず、細胞生化学的研究の場においても幅広く利用される、重要かつ汎用的な新規ツールとなりうると考えられる。
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. *
また本発明は、以上説明したように構成されているため、評価対象となる細胞を3次元的な培養環境の中の特定の位置に、正確に配置した、ハイドロゲル基材を提供することが可能となる。細胞の初期位置を制御することによって、その浸潤挙動の評価を行う際に、データのばらつきが少なくなり、信頼性が高くなるため、より正確な細胞評価を可能とするシステムとして、幅広く利用されうるものと期待される。
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. *
さらに本発明は、以上説明したように構成されているため、通常は非常に手間がかかり、さらに信頼性の低い、細胞を用いたアッセイを、より簡便に行うための手法を提供できる。特に、ハイドロゲル基材の外部に浸潤した細胞の個数をカウントするだけで、その浸潤割合を簡便に測定することが可能となるため、複雑な画像解析技術や光学系が不要なシステムを提供できる可能性が高い。そのため、コストの面において、既存のシステムよりも優位であり、細胞を用いた評価を行う産業分野や学術分野において幅広く使用される、新規細胞評価手法を提供できるものと考えられる。
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)
- ハイドロゲルによって構成され、 ファイバー状又はシート状の形態を有し、 長さ方向の軸に対して垂直な平面における断面Sの内部において、周縁部が前記断面Sの周縁部に接しておらずかつ第一の細胞が存在する領域Aを有し、 さらに前記領域Aは、前記断面Sにおける前記領域A以外の領域Bと比較して、弾性率の低いハイドロゲルによって構成されている細胞評価用ハイドロゲル基材。 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.
- 前記ハイドロゲル基材は、前記断面Sにおいて部分的に前記領域A及び前記領域Bに接し、かつ、部分的に前記断面Sの周縁部に接する領域Cを有し、 さらに前記領域Cは、前記領域Bと比較して弾性率の低いハイドロゲルによって構成されている請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 領域Bには、前記第一の細胞とは異なる第二の細胞が存在する請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 前記領域Cには、前記第二の細胞が存在する請求項2に記載の細胞評価用ハイドロゲル基材。 The hydrogel substrate for cell evaluation according to claim 2, wherein the second cell is present in the region C.
- 前記領域A、前記領域B及び領域Cを構成するハイドロゲルの少なくともいずれかは、アルギン酸又はアルギン酸の誘導体によって構成されている請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 前記領域A及び前記領域Cを構成するハイドロゲルの少なくともいずれかには、アルギン酸プロピレングリコールが含まれている請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 前記領域A、前記領域B及び領域Cを構成するハイドロゲルのマトリックス中には、細胞外マトリックスを構成するタンパク質及び細胞接着性のペプチド分子の少なくともいずれかが含まれている請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 前記第一の細胞とは、癌細胞である請求項1に記載の細胞評価用ハイドロゲル基材。 The hydrogel substrate for cell evaluation according to claim 1, wherein the first cell is a cancer cell.
- 前記第二の細胞とは、癌細胞以外の細胞である請求項1に記載の細胞評価用ハイドロゲル基材。 The hydrogel substrate for cell evaluation according to claim 1, wherein the second cell is a cell other than a cancer cell.
- 前記第二の細胞とは、繊維芽細胞、内皮細胞、上皮細胞、平滑筋細胞、骨格筋細胞、中皮細胞、実質細胞、腺細胞、表皮細胞、神経細胞、骨芽細胞、これらの細胞の前駆細胞、各種幹細胞より分化させた細胞の少なくともいずれかである請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 前記領域Aの面積は、前記第一の細胞の平均直径をDとすると、10D2以下である請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 前記領域Aの面積をSとし、前記領域Aの周囲長をLとすると、前記領域Aの円形度4πS/L2は0.5以上である請求項1に記載の細胞評価用ハイドロゲル基材。 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. .
- 前記第一の細胞の平均直径をDとすると、領域Cの幅は3D以下である請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 直径500マイクロメートル以下のファイバー状である請求項1に記載の細胞評価用ハイドロゲル基材。 The hydrogel substrate for cell evaluation according to claim 1, which is in a fiber form having a diameter of 500 μm or less.
- 厚さ500マイクロメートル以下のシート状であり、前記断面Sの内部において、前記領域Aは複数個所存在する請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 前記領域Aに含まれる、前記第一の細胞の密度は、1立方センチメートル当たり10万個以上である請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 前記領域B及び前記領域Cに含まれる、前記第二の細胞の密度は、1立方センチメートル当たり100万個以上である請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 少なくとも2つの入口I1~In(n≧2)と、前記入口I1~Inにそれぞれ接続する入口流路CI1~CInと、前記入口流路CI1~CInが同時あるいは段階的に合流する少なくとも1つの合流点P1~Pm(m≧1)と、前記合流点P1~Pmより下流に存在する合流流路Gと、前記合流流路Gの下流に存在する出口Oを有する、流路構造Xに対し、アルギン酸ナトリウムを含み、かつ前記第一の細胞を懸濁させた、第一の水溶液を、前記入口I1を介して、また、アルギン酸ナトリウムを含む第二の水溶液を、前記入口I2を介して、それぞれ連続的に導入し、 前記流路構造Xの内部において、前記第一の水溶液および前記第二の水溶液を接触させ、さらに前記流路構造Xの内部乃至外部において、前記第一の水溶液および前記第二の水溶液をゲル化剤水溶液と接触させることによって、 前記第一の水溶液及び前記第二の水溶液が接触した状態で連続的にゲル化することによって作製されたものであり、 また、前記領域Aは前記第一の水溶液がゲル化することによって形成されたものであり、かつ、前記領域Bは前記第二の水溶液がゲル化することによって形成されたものである請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 少なくとも3つの入口I1~In(n≧3)と、前記入口I1~Inにそれぞれ接続する入口流路CI1~CInと、前記入口流路CI1~CInが同時あるいは段階的に合流する少なくとも1つの合流点P1~Pm(m≧1)と、前記合流点P1~Pmより下流に存在する合流流路Gと、前記合流流路Gの下流に存在する出口Oを有する、流路構造Xに対し、アルギン酸ナトリウムを含み、かつ前記第一の細胞を懸濁させた、第一の水溶液を、前記入口I1を介して、また、アルギン酸ナトリウムを含む第二の水溶液を、前記入口I2を介して、さらに、アルギン酸ナトリウムを含む第三の水溶液を、前記入口I3を介して、それぞれ連続的に導入し、 前記流路構造Xの内部において、前記第一の水溶液、前記第二の水溶液、前記第三の水溶液を接触させ、 さらに、前記流路構造Xの内部又は外部において、前記第一の水溶液、前記第二の水溶液、および前記第三の水溶液をゲル化剤水溶液と接触させることによって、前記第一の水溶液および前記第二の水溶液が接触した状態で連続的にゲル化することによって作製されたものであり、 また、前記領域Aは前記第一の水溶液がゲル化することによって形成されたものであり、かつ、前記領域Bは前記第二の水溶液がゲル化することによって形成されたものであり、さらに、前記領域Cは前記第三の水溶液がゲル化することによって形成されたものである請求項1に記載の細胞評価用ハイドロゲル基材。 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.
- 前記第二の水溶液及び前記第三の水溶液の少なくともいずれかには、前記第二の細胞が懸濁されている請求項18に記載の細胞評価用ハイドロゲル基材。 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.
- 前記流路構造Xは、少なくとも一つの入口G1~Gn(n≧1)および、前記入口G1~Gnにそれぞれ接続し合流点P1~Pmのいずれかにおいて合流流路Gに合流する入口流路CG1~CGnを有しており、前記入口G1~Gnを介して前記ゲル化剤水溶液は流路構造Xに連続的に導入される請求項18に記載の細胞評価用ハイドロゲル基材。 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.
- 前記流路構造Xは、少なくとも一つの入口B1~Bn(n≧1)および、前記入口B1~Bnにそれぞれ接続し合流点P1~Pmのいずれかにおいて合流流路Gに合流する入口流路CB1~CBnを有しており、前記入口B1~Bnを介して、バッファー水溶液は流路構造Xに連続的に導入される請求項18に記載の細胞評価用ハイドロゲル基材。 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.
- 前記合流点P1~Pmのうち少なくとも一つの合流点において、前記第一の水溶液は、その上下左右の少なくともいずれかにおいて前記第二の水溶液と接触する請求項18に記載の細胞評価用ハイドロゲル基材。 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.
- 前記流路構造Xは、少なくとも部分的に、キャピラリー状の管によって構成されている請求項18に記載の細胞評価用ハイドロゲル基材。 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.
- 前記流路構造Xは、少なくとも部分的に、微細加工技術を用いて作製されたマイクロ流路構造によって構成されている請求項18に記載の細胞評価用ハイドロゲル基材。 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.
- 前記流路構造Xは、その幅、深さ、直径の値のうち少なくともいずれか一つが、少なくとも部分的に1ミリメートル以下である請求項18に記載の細胞評価用ハイドロゲル基材。 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.
- 前記第一の水溶液、前記第二の水溶液及び前記第三の水溶液の少なくともいずれかに含まれるアルギン酸ナトリウムの濃度は、それぞれ100mLあたり1g以下である請求項18に記載の細胞評価用ハイドロゲル基材。 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. .
- 前記第二の水溶液に含まれるアルギン酸ナトリウムの濃度は、100mLあたり1g以上である請求項18に記載の細胞評価用ハイドロゲル基材。 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.
- 前記ゲル化剤水溶液は、塩化バリウムを含む請求項18に記載の細胞評価用ハイドロゲル基材。 The hydrogel substrate for cell evaluation according to claim 18, wherein the gelling agent aqueous solution contains barium chloride.
- 前記ゲル化剤水溶液乃至前記バッファー水溶液は、増粘剤を含む請求項18に記載の細胞評価用ハイドロゲル基材。 The hydrogel substrate for cell evaluation according to claim 18, wherein the aqueous gelling agent solution or the aqueous buffer solution contains a thickener.
- 前記第一の水溶液乃至前記第三の水溶液は、アルギン酸プロピレングリコールを含む請求項18に記載の細胞評価用ハイドロゲル基材。 The hydrogel substrate for cell evaluation according to claim 18, wherein the first aqueous solution to the third aqueous solution contain propylene glycol alginate.
- 前記第一の水溶液乃至前記第三の水溶液に含まれる前記アルギン酸プロピレングリコールの濃度は、それぞれ100mLあたり1g以上である請求項31に記載の細胞評価用ハイドロゲル基材。 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.
- 請求項1に記載の細胞評価用ハイドロゲル基材を用いる細胞評価手法。 A cell evaluation method using the hydrogel substrate for cell evaluation according to claim 1.
- 前記ハイドロゲル基材内部に包埋した細胞に対し、培養操作を行い、前記第一の細胞が前記ハイドロゲル基材の内部を浸潤しゲル外部にまで達する個数あるいは割合を、計測することによって、細胞の浸潤を定量的に評価する請求項33に記載の細胞評価手法。 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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