Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
  • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/20—Material Coatings
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/22—Transparent or translucent parts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/46—Means for fastening
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/48—Holding appliances; Racks; Supports
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/02—Membranes; Filters
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/10—Hollow fibers or tubes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Definitions

• the disclosed technology relates to cell evaluation methods and evaluation devices.
• Japanese Patent Application Laid-Open No. 2012-100642 describes a cultured cell evaluation method for measuring the adhesive force between the substrate surface and the cells using the shear stress of the fluid in cells cultured on the substrate surface.
• WO 2015/125742 discloses a serpentine passage through which a liquid passes, and a plurality of cell-seeding regions in which cells passing through the passage are seeded on the bottom surface of the passage are equally spaced along the passage. describes a cell culture vessel provided with.
• endothelial cells were cultured in cell culture dishes, and endothelial cell monolayers were 14 mm wide and 0.5 mm high. Loading into a parallel plate flow chamber with a 5 mm flow section and exposure to shear stress is described.
• Adherent cells are known to exhibit various responses to the flow field, such as orientation, differentiation, detachment, and cell death.
• Cell adhesion properties are an important evaluation index in examining cell culture conditions and quality evaluation. is.
• evaluation of cell adhesiveness is also required in research and development of culture substrates and cell adhesives, and is an important concern in both research and industrial applications. Therefore, in a wide range of fields related to adherent cells, there is a demand for a technique for simply and efficiently evaluating the response of adherent cells to liquid flow.
• Patent Documents 1 and 2 when a closed microchannel is used, it is necessary to introduce cells into the closed microchannel. It is not easy to compartmentalize these cell aggregates and arrange them along the channel. In addition, there is a risk of damaging the cells when introducing the cells into the microchannel. Furthermore, the medium components may become non-uniform in the closed microchannel, creating an environment different from the standard culture environment. As a result, the state of the cells may be affected, which in turn may affect the evaluation results. In the technique of Non-Patent Document 1, a single monolayer of endothelial cells is targeted for processing, and multiple targets are not treated collectively, so efficient cell evaluation is difficult.
• the disclosed technology has been made in view of the above points, and aims to simply and efficiently evaluate the response of adhesive cells to liquid flow.
• a method for evaluating cells according to the disclosed technology includes culturing cells such that a plurality of cells or a plurality of cell aggregates are arranged on a culture surface, installing a detachable channel on the culture surface, providing a fluid flow to each of the plurality of cells or plurality of cell clumps by passing the liquid through.
• the channel may be formed by bringing the surface of a channel member having grooves forming the channel into close contact with the culture surface.
• the flow path member may be aligned so that a plurality of cells or a plurality of cell aggregates are arranged along the liquid flow direction. Alignment of the channel member may be performed using a first alignment mark provided on the surface of the channel member and a second alignment mark provided on the culture surface.
• the shear stress applied to each of the plurality of cells or the plurality of cell aggregates by the liquid flow may vary along the liquid flow direction.
• the shear stress applied to each of the plurality of cells or the plurality of cell aggregates by the liquid flow may vary linearly along the direction of flow of the liquid.
• a shear stress applied to each of the plurality of cells or the plurality of cell aggregates by the liquid flow may be constant at each position in the flow direction of the liquid.
• the area of the cross-section of the channel that intersects the direction of liquid flow may vary along the direction of liquid flow.
• the cross-sectional area of the flow channel that intersects the liquid circulation direction may increase from the upstream side toward the downstream side in the liquid circulation direction.
• the cell evaluation method according to the disclosed technology can include culturing cells so that a plurality of cells or a plurality of cell aggregates form a predetermined pattern on the culture surface.
• a cell evaluation method according to the disclosed technology includes attaching a sheet having a plurality of openings to a culture surface, and forming a plurality of cells or a plurality of cell aggregates in the exposed portions of the culture surface at the openings. obtain.
• the multiple cells or multiple cell aggregates may have the same size.
• An evaluation device is an evaluation device for applying a liquid flow to a plurality of cells or a plurality of cell aggregates arranged on a culture surface, and has a detachable flow channel on the culture surface.
• a liquid flow is provided to each of a plurality of cells or a plurality of cell aggregates by forming a channel and causing a liquid to flow through the channel.
• the evaluation device may include a channel member having grooves forming channels on its surface.
• a channel is formed by bringing the grooved surface of the channel member into close contact with the culture surface.
• the channel may be configured such that the shear stress applied to each of the plurality of cells or the plurality of cell aggregates varies along the liquid flow direction.
• the channel may be configured such that the shear stress applied to each of the plurality of cells or the plurality of cell aggregates varies linearly along the liquid flow direction.
• the area of the cross-section of the channel that intersects the direction of liquid flow may vary along the direction of liquid flow.
• the cross-sectional area of the flow channel that intersects the liquid circulation direction may increase from the upstream side toward the downstream side in the liquid circulation direction.
• An evaluation device includes an attachment section to which a channel member is attached, a holding section that rotatably or movably holds the attachment section, and a mounting area on which a substrate having a culture surface is mounted. and a fixing part for fixing the holding part at a position corresponding to the mounting area.
• the flow channel member and the mounting portion may be made of a material having optical transparency.
• a liquid flow is applied to a plurality of cells or a plurality of cell aggregates arranged on an arbitrary substrate using a detachable channel. can be performed simply and efficiently.
• FIG. 1 is a perspective view showing an example of a configuration of a masking sheet used for cell pattern culture according to an embodiment of technology disclosed herein.
• FIG. 1 is a diagram showing an example of a cell culture method according to an embodiment of technology disclosed herein;
• FIG. 1 is a diagram showing an example of a cell culture method according to an embodiment of technology disclosed herein;
• FIG. 1 is a diagram showing an example of a cell culture method according to an embodiment of technology disclosed herein;
• FIG. 1 is a diagram showing an example of a cell culture method according to an embodiment of technology disclosed herein;
• FIG. 1 is a diagram showing an example of a cell culture method according to an embodiment of technology disclosed herein;
• FIG. 1 is a diagram showing an example of a cell culture method according to an embodiment of technology disclosed herein;
• FIG. 1 is a diagram showing an example of a cell culture method according to an embodiment of technology disclosed herein;
• FIG. 1 is a diagram showing an example of a cell culture method according to an embodiment
• FIG. 1 is a diagram showing an example of a cell culture method according to an embodiment of technology disclosed herein;
• FIG. 1 is a perspective view showing an example of a configuration of a flow channel member according to an embodiment of technology disclosed herein;
• FIG. 4 is a plan view showing an example of a configuration of a flow channel member according to an embodiment of technology disclosed herein;
• FIG. 10 is a diagram showing how a channel member is installed on a culture surface.
• FIG. 10 is a diagram showing how a channel member is installed on a culture surface.
• 1 is a perspective view showing an example of the configuration of first alignment marks and second alignment marks according to an embodiment of technology disclosed herein;
• FIG. 4 is a plan view showing a state in which a shear stress is applied to a cell aggregate by a liquid flow
• FIG. 10 is a diagram showing an example of the relationship between the position in the flow direction of the liquid flowing through the channel and the shear stress caused by the liquid flow according to the embodiment of the technology disclosed herein
• FIG. 3 is a diagram showing a cross section of a flow channel according to an embodiment of technology disclosed on a coordinate plane
• FIG. 4 is a diagram showing an example of a situation in which cell aggregates are detached due to shear stress.
• FIG. 4 is a diagram showing an example of the configuration of a channel according to an embodiment of the technology disclosed herein, and the relationship between the position in the flow direction of the liquid flowing through the channel and the shear stress caused by the liquid flow;
• FIG. 4 is a diagram showing an example of the configuration of a channel according to an embodiment of the technology disclosed herein, and the relationship between the position in the flow direction of the liquid flowing through the channel and the shear stress caused by the liquid flow;
• 1 is a cross-sectional view showing an example of the configuration of an evaluation device according to an embodiment of technology disclosed herein;
• FIG. 1 is a cross-sectional view showing an example of the configuration of an evaluation device according to an embodiment of technology disclosed herein;
• FIG. 3 is a perspective view showing an example of a configuration of an attachment portion according to an embodiment of technology disclosed herein;
• FIG. FIG. 4 is a cross-sectional view showing a state in which a flow path member is attached to an attachment portion according to an embodiment of technology disclosed herein;
• FIG. 10 is a perspective view showing an example of a configuration of a holding portion holding an attachment portion according to an embodiment of technology disclosed herein;
• FIG. 10 is a plan view showing an example of a configuration of a holding portion that holds an attachment portion according to an embodiment of technology disclosed herein;
• FIG. 14C is a cross-sectional view along line 14C-14C in FIG. 14B;
• FIG. 4 is a perspective view showing an example of a configuration of a base portion that constitutes a holding portion according to an embodiment of technology disclosed herein;
• FIG. 10 is a perspective view showing an example of a configuration of a lid portion that constitutes a holding portion according to an embodiment of technology disclosed herein;
• FIG. 4 is a plan view showing an example of a configuration of a flow channel member according to an embodiment of technology disclosed herein;
• FIG. 10 is a micrograph of cell colonies patterned by pattern culture according to an embodiment of the disclosed technique;
• FIG. FIG. 10 is a diagram showing a flow channel member according to an embodiment of technology disclosed herein;
• FIG. 10 is a diagram showing the mounting portion held by the holding portion according to the embodiment of the technology disclosed;
• FIG. 2 illustrates an evaluation device according to an embodiment of the disclosed technique;
• 10 is a micrograph showing how cell colonies arranged on a culture surface according to an embodiment of the disclosed technology are detached by shear stress caused by liquid flow.
• FIG. 10 is a microphotograph showing the state of the entire channel after applying the liquid flow according to the embodiment of the disclosed technique.
• a cell evaluation method includes a first step of culturing cells on a culture surface so that a plurality of cells or a plurality of cell aggregates are arranged on the culture surface; A second step of providing a removable channel, and a third step of providing a liquid flow to each of the plurality of cells or the plurality of cell aggregates by causing liquid to flow through the channel. Details of the above-described first to third steps will be described below.
• FIG. 1 is a perspective view showing an example of the configuration of a masking sheet 10 used for pattern culture of cells.
• the masking sheet 10 is made of a material that is adhesive to the surface (culture surface) of a substrate (not shown) used for adherent culture of cells.
• the masking sheet 10 preferably has sufficient flexibility to ensure adhesion to the culture surface.
• rubber members such as silicone rubber (VMQ, PVMQ, FVMQ), isoprene rubber (IR) and fluororubber (FKM) can be preferably used.
• Masking sheet 10 has a plurality of openings 12 extending through the substrate. The number, shape and size of openings 12 and the spacing between adjacent openings 12 can be arbitrarily determined.
• FIG. 1 illustrates a configuration in which a plurality of rectangular openings 12 having the same size are arranged in a straight line.
• FIG. 2A to 2G are diagrams showing an example of a cell culture method for performing pattern culture using the masking sheet 10.
• FIG. 1A a substrate 20 and a masking sheet 10 are prepared (FIG. 2A).
• the surface of the substrate 20 constitutes the culture surface.
• a commercially available cell culture dish can be used as the substrate 20 .
• the masking sheet 10 may be cut into the same shape as the substrate 20 (circular in the example shown in FIG. 2A).
• the diameter of masking sheet 10 is preferably about the same as the diameter of substrate 20 .
• the masking sheet 10 is attached to the surface (culture surface) of the substrate 20 (Fig. 2B). Specifically, after bringing the masking sheet 10 into contact with the surface (culture surface) of the substrate 20 , the masking sheet 10 is brought into close contact with the substrate 20 by applying a load to the masking sheet 10 .
• the flexibility of the masking sheet 10 ensures high adhesion, and the masking sheet 10 can be attached to the substrate 20 without generating air bubbles between the masking sheet 10 and the substrate 20 .
• the surface (culture surface) of the substrate 20 is exposed at the openings 12 of the masking sheet 10 .
• a cell adhesive 30 is applied to the surface (culture surface) of the substrate 20 (Fig. 2C).
• the cell adhesive 30 adheres to the surface (culture surface) of the substrate 20 exposed at the openings 12 of the masking sheet 10 .
• a pattern of the cell adhesive 30 corresponding to the pattern of the openings 12 of the masking sheet 10 is formed on the culture surface.
• Cell adhesives 30 can be extracellular matrix proteins such as vitronectin, laminin, fibronectin, collagens and gelatins.
• the masking sheet 10 is peeled off from the culture surface (Fig. 2D).
• a patterned cell adhesive 30 is maintained on the surface (culture surface) of the substrate 20 .
• a blocking agent is applied to the surface (culture surface) of the substrate 20 .
• This treatment is a blocking treatment for inhibiting non-specific cell adhesion in areas other than the area to which the cell adhesive 30 adheres on the culture surface.
• a blocking agent for example, a bovine serum albumin solution prepared to a predetermined concentration can be used.
• the cells 40 may be adhesive cells, such as iPS cells (induced pluripotent stem cells), ES cells (Embryonic Stem Cells), stem cells such as mesenchymal stem cells and hematopoietic stem cells, lung cells, gastric cells, hepatocytes.
• iPS cells induced pluripotent stem cells
• ES cells Embryonic Stem Cells
• stem cells such as mesenchymal stem cells and hematopoietic stem cells, lung cells, gastric cells, hepatocytes.
• somatic cells such as kidney cells, intestinal cells, nerve cells, eye cells, vascular endothelial cells, cardiomyocytes and bone cells, lung cancer cells, stomach cancer cells, colon cancer cells, breast cancer cells, leiomyosarcoma cells, fibrosarcoma cells and Cancer cells such as osteosarcoma cells and established cell lines such as HEK293 cells (human embryonic kidney cells) and Vero cells (African green monkey kidney cells) can be used.
• Cells can be used.
• Cells derived from animals other than humans specifically, mice, rats, rabbits, pigs, dogs, monkeys, cows, horses, sheep, chickens, etc. may also be used.
• the masking sheet 10 is peeled off from the culture surface after the cell adhesive 30 is applied and before the cells 40 are seeded. and the masking sheet 10 may be peeled off from the culture surface after the cells 40 are seeded.
• detachable channels are provided on the culture surface on which the cells or cell aggregates are arranged by the pattern culture described above.
• 3A and 3B are a perspective view and a plan view, respectively, showing an example of the configuration of a channel member 50 for forming a channel that can be attached to and detached from the culture surface.
• the outer shape of the channel member 50 is not particularly limited, it preferably has a shape corresponding to the shape of the culture surface.
• FIG. 3A illustrates a cylindrical channel member 50 assuming that cell culture is performed using a commercially available dish having a circular culture surface.
• a groove 51 forming a flow path is provided on one surface of the flow path member 50 .
• An inlet 52 is connected to one end of the groove 51 and an outlet 53 is connected to the other end of the groove 51 .
• the inflow port 52 and the outflow port 53 are led out to the surface of the channel member 50 opposite to the surface on which the groove 51 is formed.
• the channel member 50 is preferably made of a material that is easy to process and has high adhesion to the culture surface.
• the flow path member 50 is made of a material having optical transparency.
• PDMS polydimethylsiloxane
• the channel member 50 can be formed, for example, by casting.
• FIG. 4A and 4B are cross-sectional views showing how the channel member 50 is installed on the culture surface S1 of the substrate 20 on which the cell aggregates 40a are arranged.
• a channel 55 is formed on the culture surface S1 by bringing the surface of the channel member 50 formed with the grooves 51 into close contact with the culture surface S1.
• the channel 55 may be a micro channel whose height and width are on the order of hundreds of meters.
• the channel member 50 can be easily removed from the culture surface S1, and can be repeatedly attached to and detached from the culture surface S1.
• the plurality of cell aggregates 40a formed on the culture surface S1 are aligned along the direction of flow of the liquid flowing through the channel 55 (that is, the direction of the channel).
• the flow channel members 50 are aligned so as to be aligned.
• alignment is performed so that each of the plurality of cell aggregates 40a is positioned on the centerline of the channel 55 in the width direction. Since the channel member 50 is made of a light-transmitting material, the positions of both the grooves 51 and the cell aggregates 40a can be visually recognized through the channel member 50. Therefore, the position of the channel member 50 Alignment becomes easier.
• FIG. 5 is a perspective view showing an example of the configuration of the first alignment mark 201 and the second alignment mark 202 used for alignment of the channel member 50.
• the first alignment mark 201 is provided on the surface of the channel member 50 on which the grooves 51 are formed.
• the first alignment mark 201 has a convex pattern protruding from the surface of the channel member 50 on which the grooves 51 are formed.
• a second alignment mark 202 is provided on the culture surface.
• the second alignment mark 202 has a concave pattern corresponding to the first alignment mark 201 .
• the flow channel member 50 is aligned so that the first convex alignment mark 201 is fitted into the second concave alignment mark 202 .
• FIG. 5 exemplifies a cross-shaped pattern as the pattern of the first alignment mark 201 and the second alignment mark 202, any pattern can be used.
• a second alignment mark 202 may be formed on the masking sheet 10 . That is, the masking sheet 10 may have not only the openings 12 (see FIG. 1) for patterning cells or cell aggregates, but also openings that constitute the second alignment marks 202 . In this case, a perforation line is provided between the opening 12 of the masking sheet 10 and the alignment mark 202, and in the pattern culture, the part where the opening 12 of the masking sheet 10 is formed is cut along the perforation line, whereby the second The second alignment mark 202 formation portion may be left on the culture surface while the second alignment mark 202 formation portion is separated from the second alignment mark 202 formation portion.
• the relative positional relationship between the openings 12 for patterning cells or cell aggregates and the second alignment marks 202 is fixed.
• aligning the path members 50 it is possible to match the arrangement of the channels 55 with the cells or cell aggregates patterned on the culture surface. Further, alignment of the flow path member 50 can be easily performed by using an evaluation device 100 (see FIGS. 12A and 12B), which will be described later.
• the case of aligning the flow path member 50 using a combination of the first convex alignment mark 201 and the second concave alignment mark 202 is illustrated.
• the alignment of the flow path member 50 may be performed using a combination of the alignment mark and the concave second alignment mark.
• the combination of the first convex alignment mark 201 and the second concave alignment mark, and the combination of the first concave alignment mark and the second concave alignment mark are used to form the flow channel member 50.
• Alignment may be performed. In this case, rough alignment is first performed using a combination of the first convex alignment mark and the second concave alignment mark, and then the first concave alignment mark and the second concave alignment mark are used.
• a combination of marks may be used to provide precise alignment.
• liquid is introduced from the inflow port 52 of the channel member 50 using a liquid sending means such as a pump, and the liquid is circulated through the channel 55, whereby the plurality of cells arranged on the culture surface S1 are A fluid flow is applied to each of the cells or multiple cell clumps.
• a case of evaluating the response of cell aggregates when shear stress is applied to each of a plurality of cell aggregates by liquid flow is exemplified. Not limited to shear stress.
• water pressure i.e., pressure acting on the cell surface in the direction of the cell center
• pressure drag i.e., force in the direction of flow acting on the surface of the cell perpendicular to the direction of liquid flow
• medium concentration i.e., cell detachment agent concentration, etc.
• cell detachment agent concentration may be parameters that act on cells or cell aggregates.
• FIG. 6 shows how shear stress is applied to each of the plurality of cell aggregates 40a arranged on the culture surface by causing the liquid to flow through the channels 55 formed on the culture surface. It is a top view. A plurality of cell aggregates 40a are arranged along the liquid flow direction indicated by the arrow in FIG.
• FIG. 7 is a diagram showing an example of the relationship between the position in the flow direction of the liquid flowing through the channel 55 and the shear stress caused by the liquid flow.
• a plurality of plots in FIG. 7 correspond to each of the cell aggregates 40a arranged on the culture surface.
• the shear stress applied to each of the plurality of cell aggregates 40a changes linearly along the direction of liquid flow. More specifically, the shear stress decreases linearly from the inlet 52 side (upstream side) of the channel 55 toward the outlet 53 side (downstream side).
• the change in shear stress along the liquid flow direction is defined by the cross-sectional area (hereinafter referred to as the cross-sectional area of the flow channel) of the flow channel 55 that intersects the flow direction of the liquid. It is realized by changing along
• the cross-sectional shape of the channel is rectangular, and the height of the channel 55 is constant over the entire area.
• the width of the flow path 55 continuously increases from the upstream side toward the downstream side in the liquid flow direction. That is, the cross-sectional area of the flow channel continuously expands from the upstream side toward the downstream side in the liquid flow direction.
• Equation (1) The relationship between the shear stress ⁇ [Pa] caused by the liquid flow and the width w [m] of the channel 55 is expressed by the following equation (1) when the channel width is sufficiently large relative to the channel height.
• ⁇ is the viscosity [Pa ⁇ s] of the liquid flowing through the channel 55 and Q is the flow rate [m 3 /sec] of the liquid flowing through the channel 55 .
• x is the position [m] in the flow direction of the liquid flowing through the channel 55 and h is the height [m] of the channel 55 .
• the cross-sectional shape of the flow path 55 is rectangular.
• equation (1) is applicable when the width w is sufficiently large relative to the height h.
• the quantity becomes large.
• the total area of the flow paths becomes large, resulting in a decrease in area efficiency.
• the width w is sufficiently large relative to the height h means that the width w is ten times or more the height h, for example.
• the flow path is designed using an exact solution of shear stress that can be applied even if the ratio of width w to height h is small.
• the left side of FIG. 8 is a view showing the cross section of the flow channel 55 having a rectangular cross section with a width w [m] and a height h [m] on a coordinate plane, and the right side of FIG.
• the flow rate Q and the height h of the channel 55 are fixed to arbitrary values, and the width w of the channel 55 and the width w
• the width w of the channel 55 and the width w By numerically obtaining the relationship w(x) with the position x, as shown in FIG. 7, a channel 55 is constructed in which the shear stress caused by the liquid flow changes linearly along the liquid flow direction. can do.
• the cell aggregates 40a are separated from the culture surface.
• some cell aggregates 40a located on the inlet side (upstream side) of the channel 55 where a relatively large shear stress acts are It is assumed that some cell aggregates 40a located on the outflow port side (downstream side) of the channel 55, which are peeled off from the culture surface and acted on by a relatively small shear stress, remain on the culture surface without being peeled off. be. In this case, it is possible to estimate the shear stress threshold at which the cell aggregate 40a is detached from the boundary position between the region R1 where detachment occurs and the region R2 where detachment does not occur.
• the outline of the side surface of the flow path 55 in plan view may be linear, and the width w of the flow path 55 may be changed linearly along the direction of liquid flow. According to this configuration, while the design of the flow path is facilitated, the change in the magnitude of the shear stress with respect to the position in the flow direction of the liquid becomes non-linear. The change in shear stress with respect to the displacement of
• the channel cross-sectional area (the width w of the channel 55) may be constant throughout the liquid flow direction.
• the shear stress applied to each of the cell aggregates 40a can be made constant at each position in the liquid flow direction. According to this configuration, it is possible to evaluate the variation in the adhesive strength of the plurality of cell aggregates 40a, for example.
• the evaluation device 100 has a function of supporting the installation of the channel member 50 on the culture surface S1.
• the evaluation device 100 includes a channel member 50 , a mounting portion 70 , a holding portion 80 and a fixing portion 90 . The details of each of the components of the evaluation device 100 will be described below.
• FIG. 13A and 13B are a perspective view and a plan view, respectively, showing an example of the configuration of the attachment portion 70.
• FIG. 13C is a cross-sectional view along line 13C-13C in FIG. 13B.
• 13D is a cross-sectional view showing a state in which the flow path member 50 is attached to the attachment portion 70.
• the mounting portion 70 has a disk-shaped flange portion 71 and a disk-shaped projecting portion 72 having a smaller diameter than the flange portion 71 . As shown in FIG. 13D, the channel member 50 is attached to the surface of the projection 72 . The flow path member 50 is fixed to the mounting portion 70 by its close contact. The flow path member 50 can be easily detached from the mounting portion 70 and can be repeatedly attached to and detached from the mounting portion 70 .
• the attachment portion 70 has a through hole 73 that communicates with the inlet 52 and the outlet 53 of the flow path member 50 when the flow path member 50 is attached.
• the mounting portion 70 be made of a material having high adhesion to the flow path member 50 .
• the mounting portion 70 be made of a material having optical transparency. By configuring the mounting portion 70 with a material having light transmittance, alignment when mounting the flow path member 50 to the mounting portion 70 is facilitated. Polycarbonate can be suitably used as the material of the mounting portion 70 .
• the holding part 80 holds the mounting part 70 rotatably or movably.
• 14A and 14B are a perspective view and a plan view, respectively, showing an example of the configuration of the holding portion 80 holding the mounting portion 70.
• FIG. FIG. 14C is a cross-sectional view along line 14C-14C in FIG. 14B.
• 15A is a perspective view showing an example of the configuration of a base portion 81 that constitutes the holding portion 80
• FIG. 15B is a perspective view showing an example of the configuration of a lid portion 82 that constitutes the holding portion 80.
• the base portion 81 has a recessed portion 83 into which the flange portion 71 of the mounting portion 70 is fitted, and an opening portion 84 through which the projecting portion 72 of the mounting portion 70 is inserted when the flange portion 71 is fitted into the recessed portion 83 .
• the flange portion 71 is sandwiched between the base portion 81 and the lid portion 82 .
• An opening 85 is provided in the center of the lid portion 82 , and the surface of the flange portion 71 is exposed at the opening 85 .
• Two end surfaces of the base portion 81 facing each other are provided with notch portions 86 respectively, and the outer peripheral portion of the flange portion 71 protrudes outside the holding portion 80 beyond the notch portions 86 .
• a clearance C on the order of several hundred ⁇ m is provided between the side surface of the concave portion 83 provided in the base portion 81 and the flange portion 71, and the mounting portion 70 rotates while being held by the holding portion 80. It is rotatable around the axis AX and is movable in the planar direction of the culture surface (XY direction in FIG. 14B). Rotation and movement of the mounting portion 70 can be performed by manually operating the outer peripheral portion of the flange portion 71 protruding outside the holding portion 80 . By holding the attachment part 70 rotatably or movably, the channel member 50 can be aligned with the culture surface in a state where the channel member 50 is attached to the attachment part 70 .
• the fixing part 90 has a mounting area R3 on which the substrate 20 having the culture surface is mounted.
• a groove 91 having a shape corresponding to the outer shape of the substrate 20 is provided in the mounting region R3.
• An opening 92 is provided in the mounting area R3, and the culture surface S1 can be observed from the bottom side of the fixing part 90 through the opening 92.
• the fixing portion 90 fixes the holding portion 80 at a position corresponding to the mounting region R3. That is, the relative positional relationship between the mounting area R3 and the holding portion 80 is fixed.
• the holding portion 80 may be fastened to the fixed portion 90 using bolts 93, for example.
• the holding portion 80 With the flow path member 50 attached to the mounting portion 70, the mounting portion 70 held by the holding portion 80, and the substrate 20 mounted on the mounting region R3 of the fixing portion 90 (see FIG. 12A), the holding portion 80 is attached to the fixing portion 90. , the channel member 50 is installed on the culture surface S1, and a channel 55 is formed on the culture surface S1 (see FIG. 12B).
• the fixed part 90 has a stopper 94 including a spring, and the holding part 80 is supported by the stopper 94 .
• the flow path member 50 and the culture surface S1 can be maintained in a non-contact state. can be aligned.
• the mounting portion 70 is held by the holding portion 80 .
• the flow path member 50 is attached to the attachment portion 70 .
• the grooves 51, the inlets 52, the outlets 53, and the through holes 73 of the mounting portion 70 of the channel member 50 are filled with the liquid.
• the liquid is fed through the tube inserted into the through-hole 73, whereby the grooves 51, the inlets 52, the outlets 53, and the through-holes 73 are can be filled with liquid.
• the substrate 20 on which the cells or cell aggregates 40a are arranged on the culture surface S1 is mounted on the mounting region R3 of the fixing portion 90. As shown in FIG.
• the holding part 80 is attached to the fixing part 90 .
• the bolts 93 for fastening the holding portion 80 to the fixed portion 90 are temporarily tightened.
• the holding part 80 is supported by the stopper 94, and the channel member 50 and the culture surface S1 are maintained in a non-contact state.
• the flow channel member 50 is aligned by rotating and moving the holding portion 80 .
• the channel member 50 is aligned so that the plurality of cell aggregates 40a formed on the culture surface S1 are arranged along the flow direction of the liquid flowing through the channel 55.
• FIG. The relative positional relationship between groove 51 and cell aggregate 40 a can be confirmed through substrate 20 exposed from opening 92 of fixing portion 90 .
• Alignment of the channel member 50 may be performed using a microscope (not shown) installed below the fixed portion 90 . Further, the alignment of the flow path member 50 may be performed using the first alignment mark 201 and the second alignment mark 202 as illustrated in FIG. After the alignment of the flow channel member 50 is completed, the bolts 93 are fully tightened so that the surface of the flow channel member 50 on which the grooves 51 are formed is brought into close contact with the culture surface S1. Thereby, the channel 55 is formed on the culture surface S1.
• the liquid is sent through the tube 110 (see FIG. 12B) inserted into the through-hole 73 of the mounting portion 70, and the liquid is circulated in the flow path 55, whereby each of the plurality of cell aggregates 40a is caused to flow by the liquid flow.
• Apply shear stress The response of cell aggregate 40 a to shear stress can be observed through substrate 20 exposed from opening 92 of fixing part 90 . Observation of the response of the cell aggregate 40 a may be performed using a microscope (not shown) installed below the fixing part 90 .
• the method for evaluating cells includes culturing cells on a culture surface such that a plurality of cells or a plurality of cell aggregates are arranged on the culture surface, and and providing a fluid flow to each of the plurality of cells or the plurality of cell aggregates by providing a removable channel 55 in the channel 55 and causing a liquid to flow through the channel 55 .
• the channel 55 is formed by bringing the surface of the channel member 50 having the grooves 51 in close contact with the culture surface.
• the flow path member 50 is aligned so that a plurality of cells or a plurality of cell aggregates are arranged along the liquid flow direction.
• a flow channel that is detachably installed on the culture surface is used, so cells cultured in an open system using a commercially available dish can be evaluated. can be done. That is, it can be cultured in a standard environment, and it is easy to compartmentalize a plurality of cells or a plurality of cell aggregates and arrange them regularly. It is also easy to quantify or normalize the state of cells in each compartment. In addition, damage to cells during culture can be suppressed as compared with the case of using a closed microchannel.
• the evaluation method according to the embodiments of the disclosed technology it is possible to simultaneously apply a liquid flow to a plurality of cells or a plurality of cell aggregates arranged on a culture surface, so that efficient evaluation can be performed. can be done.
• the cell evaluation method according to the embodiment of the disclosed technology it is possible to easily and efficiently evaluate the response of adhesive cells to liquid flow.
• the evaluation method according to the embodiment of the disclosed technology includes making the sizes of the plurality of cells or the plurality of cell aggregates the same. This makes it possible to quantify or normalize the states of a plurality of cells or a plurality of cell aggregates, contributing to the acquisition of useful evaluation data.
• the evaluation method according to the embodiment of the disclosed technique may include changing the shear stress applied to each of the plurality of cells or the plurality of cell aggregates by the liquid flow along the liquid flow direction. This makes it possible to apply shear stresses of different magnitudes to a plurality of cells or a plurality of cell aggregates arranged on the culture surface. It is possible to efficiently perform evaluation such as
• the change in shear stress along the liquid flow direction can be linear.
• the change in the magnitude of the shear stress along the flow direction of the liquid is non-linear, it is possible to suppress the change in the shear stress due to the displacement in the flow direction of the liquid.
• the evaluation method includes making the shear stress applied to each of the plurality of cells or the plurality of cell aggregates due to the liquid flow constant at each position in the liquid flow direction. This makes it possible to evaluate variations in responsiveness to liquid flow among a plurality of cells or a plurality of cell aggregates.
• FIG. 16 is a plan view showing an example of the configuration of the flow channel member 50 according to another embodiment of the technology disclosed.
• the channel member 50 may have a plurality of grooves 51 forming channels. As a result, it is possible to form a plurality of channels on the culture surface, and a large number of cells can be evaluated with a single liquid transfer.
• a plurality of grooves 51 may be arranged radially and a common inlet 52 may be arranged at the center.
• a masking sheet was attached to the surface of a commercially available 35 mm dish (manufactured by Falcon). A masking sheet was used in which 21 square openings each having a side length of 200 ⁇ m were arranged in a straight line. After bringing the masking sheet into contact with the surface of the dish, the masking sheet was brought into close contact with the surface of the dish by applying a load to the masking sheet.
• a 2.5 ⁇ g/ml vitronectin solution which is a cell adhesive
• a pattern of the cell adhesive was formed on the surface of the dish corresponding to the pattern of the openings of the masking sheet.
• the masking sheet was peeled off from the surface of the dish.
• a 3% bovine serum albumin solution which is a blocking agent, was applied to the surface of the dish.
• This treatment is a blocking treatment for inhibiting non-specific cell adhesion in areas other than the area to which the cell adhesive adheres on the culture surface.
• the treatment time was 30 minutes.
• the blocking agent was replaced with a phosphate buffer, and the phosphate buffer was aspirated.
• FIG. 17 is a micrograph of cell colonies patterned by the pattern culture described above. It was confirmed that a cell pattern corresponding to the pattern of the openings of the masking sheet was formed.
• a channel member was produced by processing PDMS by a casting method. Specifically, PDMS in which the ratio of the main agent and the curing agent was 10:1 was poured into a mold and cured in an environment of 50° C. for 3 hours. The mold-releasing PDMS was punched out with a hole punch to prepare a flow path member made of PDMS with a diameter of 32 mm and a height of 6 mm.
• the channel (groove) was designed so that the shear stress generated in the channel by the liquid flow changed linearly along the liquid flow direction.
• the channel had a rectangular cross-sectional shape and a constant height of 200 ⁇ m.
• the width of the channel was derived based on the rigorous solution obtained by solving the differential equation of the Navier-Stokes equation, which is the basic fluid equation, with boundary conditions for the rectangular cross-section channel.
• the width of the flow channel at the inlet side end was set to 300 ⁇ m, and the width at the outlet side end was set to 2500 ⁇ m.
• FIG. 18 shows the fabricated flow path member.
• the flow path member was attached to an attachment portion made of polycarbonate having optical transparency.
• the holding portion consisted of a base portion and a lid portion made of alumite-treated aluminum, and the mounting portion was held by sandwiching the mounting portion between the base portion and the lid portion.
• the mounting portion is capable of rotating and moving in the planar direction while being held by the holding portion.
• FIG. 19 shows the mounting portion held by the holding portion.
• the fixed part was placed in an incubator maintained in an environment of 37° C. and 5% CO 2 concentration.
• a dish with arrayed cell colonies was mounted on the mounting area of the fixing part.
• the holding part holding the mounting part to which the channel member was attached was attached to the fixed part. While observing the position of the cell pattern and the channel (groove formed in the channel member) with a microscope, the channel member was aligned so that the cell colonies on the dish were arranged along the channel.
• the holding portion was fastened to the fixing portion using bolts inserted through the screw holes provided at the four corners of the holding portion to construct the device for evaluation.
• FIG. 20 shows the built evaluation device.
• Shear stress was applied to the cell colonies arranged on the culture surface by introducing the culture medium from the inlet of the channel member using a syringe pump and circulating the liquid in the channel.
• the flow rate of the liquid flowing through the channel was set to 0.6 ml/min.
• the shear stress generated at the end of the channel on the inlet side is about 5 Pa
• the shear stress generated at the end on the outlet side of the channel is about 0.5 Pa.
• FIG. 21 is a micrograph showing how cell colonies arranged on a culture surface are detached by shear stress caused by liquid flow. As shown in FIG. 21, it was observed that the cell colonies were detached in order from the upstream side of the channel where the shear stress was relatively large.
• FIG. 22 is a microphotograph showing the state of the entire channel after the liquid flow is applied. As shown in FIG. 22, a plurality of cell colonies located downstream of the flow path with relatively low shear stress were maintained in a state of being adhered to the culture surface without detachment. It was estimated that 1.2 Pa was the shear stress threshold at which the cell colony detached from the boundary position between the region where the cell colony detached and the region where the detachment did not occur.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Sustainable Development (AREA)
• Clinical Laboratory Science (AREA)
• Immunology (AREA)
• Analytical Chemistry (AREA)
• Cell Biology (AREA)
• Molecular Biology (AREA)
• Mechanical Engineering (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Related Child Applications (1)

Publications (1)

Family

ID=87564147

Family Applications (1)

Country Status (4)

Citations (6)

• 2022
  • 2022-11-30 JP JP2023580083A patent/JPWO2023153058A1/ja active Pending
  • 2022-11-30 EP EP22925247.3A patent/EP4461815A1/en active Pending
  • 2022-11-30 WO PCT/JP2022/044238 patent/WO2023153058A1/ja not_active Ceased
• 2024
  • 2024-07-25 US US18/783,420 patent/US20240376422A1/en active Pending

Patent Citations (6)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events