JP2020028270A - Cell migration assay device - Google Patents

Abstract

To provide an effective micro fluid device for a cell migration assay without depending on a photolithography technique.SOLUTION: A micro fluid device for a cell migration assay has a first flow channel into which a migrating cell is introduced and a second flow channel into which a chemoattractant is introduced in a silicon rubber substrate incorporating a porous membrane therein, and the first flow channel and the second flow channel are partitioned by at least one porous membrane.SELECTED DRAWING: None

Description

  The present invention relates to a cell assay device. The invention particularly relates to devices for assaying cell migration.

  Cell migration is a general term for a phenomenon in which cells move according to a concentration gradient of a chemical substance (a chemoattractant such as a chemokine) or move while decomposing an extracellular matrix. These phenomena are involved in immune reactions and cancer metastasis.

  For example, in inflammatory skin tissue, macrophages present in the interstitium secrete cytokines and chemokines, which increase vascular permeability, and chemokines cause neutrophils to migrate from blood vessels to the stroma. Induces and promotes inflammation. In addition, cancer cells express chemokine receptors on the cell surface, and these cancer cells are attracted by chemokines to migrate out of blood vessels, leading to invasion of other tissues and formation of metastatic foci. obtain.

  As a system for experimentally evaluating cell migration, a 2D Gap Closure assay for observing migration on a substrate and a Boyden Chamber assay for observing migration through a porous membrane are known. However, these assays have a large deviation from actual vascular tissue and the like in that migration of cells is assayed in a large assay container containing a volume of several mL. In addition, there is a disadvantage that the migration result is only evaluated at the endpoint instead of observing the cell migration in real time.

  From around 2000, a system began to be reported using a microchannel produced by microfabrication as an analysis site for cell migration (for example, Non-Patent Document 1). The use of a microchannel enables analysis using a small amount of reagents and cells, analysis under a complicated concentration gradient, and also enables real-time observation.

  The conventional microfluidic device for cell migration analysis exemplified in Non-Patent Document 1 has been manufactured by photolithography technology. That is, a photoresist is applied to a silicon substrate, and then covered with a photomask, exposed to ultraviolet light, and developed to form a mold. A prepolymer of polydimethylsiloxane (PDMS) is poured into the mold and cured. A microchannel has been produced by removing the mold-molded and cured PDMS from the mold and bonding the surface having the channel mold to a flat glass substrate. A pseudo-vascular wall having a gap through which cells can be formed by forming a large number of square column-shaped structures in a row so as to traverse the center of the flow channel at a narrow interval of about 3 to 15 μm by the design of a photomask. Was formed. That is, one side of the flow path is a pseudo-interstitial and is filled with a chemoattractant, and the other side of the flow path separating the column rows is a pseudo-vessel lumen through which cells flow.

K. C. Chaw et al., Biomed. Microdev. (2007) 9: 597-602

  Fabrication of microfluidic devices for cell migration analysis by photolithography technology can create a precise channel structure that mimics real tissue, but requires expensive and large-scale equipment such as clean room equipment and mask aligners. It is also necessary to use expensive consumables such as photoresist and the like. At present, it is not easy for biological researchers focusing on biological experiments of cell migration to obtain or use such expensive and highly specialized engineering equipment and supplies.

  By using a microfluidic device in which an artificial porous membrane is incorporated in a silicone rubber substrate, the present inventor can perform a cell migration assay that mimics the in vivo environment extremely simply and without using photolithography. And completed the present invention.

The present invention includes the following embodiments.
[1]
In a silicone rubber substrate having a porous membrane incorporated therein, the substrate has a first channel into which migratory cells are introduced and a second channel into which a chemoattractant is introduced. A microfluidic device for cell migration assay, wherein the second channel is separated from the second channel by at least one porous membrane.
[2]
At least one of the porous membranes is incorporated into the substrate by being inserted into a notch formed at an angle of 45 ° to 90 ° with respect to a horizontal plane in the substrate; [1] The microfluidic device for cell migration assay as described above.
[3]
The microfluidic device for cell migration assay according to [1] or [2], wherein the average pore size of at least one of the porous membranes is 3 to 15 μm.
[4]
A third flow path disposed between the first flow path and the second flow path, wherein the first flow path and the third flow path are formed by a first porous membrane; The microfluidic device for cell migration assay according to any one of [1] to [3], wherein the microfluidic device is separated and the second flow path and the third flow path are separated by a second porous membrane.
[5]
The microfluidic device for cell migration assay according to any one of [1] to [4], wherein the silicone rubber contains polydimethylsiloxane.
[6]
The microfluidic device for cell migration assay according to any one of [1] to [5] is tested for cell migration in which cells introduced into the first channel permeate at least one of the porous membranes. Wherein the cell migration is cell migration dependent on the presence of a chemoattractant introduced into the second channel.

  According to the embodiment of the present invention, a drug evaluation test of a cell migration inhibitor (for example, an anti-inflammatory agent and an anti-cancer agent, particularly a cancer metastasis inhibitor) in an environment close to a living body is performed extremely inexpensively and simply without using photolithography. And so on. The device according to the present embodiment supports a cell migration evaluation test in the presence of a matrix gel or a biomolecule coating easily, similarly to or more than a device manufactured by photolithography technology. It is also possible to culture living cells such as endothelial cells on a membrane and perform an evaluation test in consideration of the interaction between the cells.

  The device is applicable to any cell type, chemoattractant, and inhibitor. Since the number of cells required for an assay by the present device is small, an evaluation test using, for example, immune cells derived from a patient can be performed.

  Compared to devices using photolithography technology, this device does not require much cost, labor and time to change the number, position, pore size, pore density, etc. Even for biologists who have difficulty using the equipment, the effects of these structural parameters on cell migration can be very easily assessed. That is, the present invention opens the opportunity to many researchers who have not been able to easily carry out cell migration experiments based on microdevices, and has elucidated the biological clarification of cell migration and the use of related drugs. Has the potential to advance research rapidly.

FIG. 1 shows an outline of a manufacturing process of a microfluidic device for cell migration assay according to one embodiment (a), and a photograph (b) of an example of the manufactured microfluidic device for cell migration assay taken from above. FIG. 2 shows an experiment in which migratory cells are accommodated in the upper channel of the three-channel device shown in FIG. 1 and the chemoattractant fMLP is introduced (right) or not (left) in the lower channel. The fluorescence microscope images at 0 hours (upper) and 3 hours (lower) from the start are shown. The lower channel is not included in the image. Individual cells are fluorescently labeled and are observed as numerous white dots. The dotted line is drawn in this drawing to indicate the position of the porous membrane, and is not an actual microscope image. FIG. 3a shows the result of measuring the number of migrating cells detected in the middle channel in the experiment of FIG. FIG. 3b shows the results of comparing the number of migrating cells after 3 hours when different concentrations of fMLP were introduced into the lower channel. FIG. 3c shows the results of measuring the number of migrating cells when different concentrations of Wortmannin were introduced into all the channels in addition to 100 nM fMLP in the lower channel. FIG. 4 shows an outline of a cell migration assay using a microfluidic device for cell migration assay according to one embodiment. In the upper channel, round cells represent migrating cells, and diamond cells represent vascular endothelial cells.

  In the present disclosure, terms such as “up”, “down”, “first”, and “second” are used for convenience in distinguishing a plurality of elements or steps, and are not necessarily absolute positional relations or orders. Is not intended to be specified. For example, two flow paths located on the same horizontal plane may be referred to as an upper flow path and a lower flow path. In this case, even if the names of the upper flow path and the lower flow path are interchanged, there may be no structural difference.

  In one aspect, the present disclosure has a first flow path and a second flow path in a silicone rubber substrate in which a porous membrane is incorporated, and the first flow path and the second flow path are separated from each other. Provided is a microfluidic device for cell migration assay, which is separated by at least one porous membrane. The microfluidic device according to the present disclosure uses silicone rubber as a device body, that is, a substrate. The silicone rubber in the present disclosure typically contains polydimethylsiloxane, and may be made of polydimethylsiloxane. At least a part of the methyl group may be replaced with another substituent such as a vinyl group or a phenyl group.

  The substrate usually has a substantially flat outer shape. The flat plate preferably has a size of not more than 25 mm in length and not more than 75 mm in width, and more preferably not more than 20 mm in length and not more than 30 mm in width. The terms "vertical" and "horizontal" are not intended to limit the flat plate to a rectangular flat plate. The thickness of the flat plate is preferably within 20 mm, more preferably within 10 mm. With these dimensions, observation of cell migration with a microscope is facilitated.

  The microfluidic device for cell migration assay of the present embodiment has at least two flow paths, that is, a first flow path and a second flow path. As is commonly understood by those skilled in the art, a channel in a microfluidic device is a space formed inside a substrate that can be filled with a microliter volume of liquid from outside (usually through a port). is there. During the execution of the cell migration assay, the fluid in the flow channel can be kept flowing or can be retained. The channel may be filled with a gel-like substance instead of a liquid. The first flow path and the second flow path are separated by at least one porous membrane. That is, the cells or liquids placed in the first flow path cannot reach the second flow path unless they pass through at least one porous membrane.

  Since the porous membrane itself is passively sandwiched and may or may not actively form a crosslink with the silicone rubber, the material of the porous membrane is not essentially limited. The porous membrane is typically made of a synthetic resin. Examples of synthetic resins include polyester, polycarbonate, polyacrylonitrile, ethylene-vinyl alcohol copolymer, polyolefin, cyclic polyolefin, polyvinyl chloride, polystyrene, polyvinyl alcohol, polyamide and the like, and mixtures thereof. Not limited. It is particularly preferred that the porous membrane comprises polyester or polycarbonate, or that the porous membrane comprises polyester or polycarbonate. The polyester is preferably polyethylene terephthalate (PET).

  The pore diameter of at least a part of the pores on the porous membrane is a size that allows permeation of the migrating cells, and the average pore diameter of the porous membrane is preferably 3 to 15 μm, more preferably 5 to 15 μm, and still more preferably 5 to 10 μm. It is. In the present disclosure, the pore diameter of each pore of the porous membrane can be defined as the diameter of a circle having the same area as the pore. The average pore diameter can be obtained by taking an image of the surface of the porous membrane with an electron microscope, measuring a plurality of (for example, at least 10, preferably at least 100) pore diameters, and averaging them. For example, the bottom of a commercially available cell culture insert is porous, and a porous membrane obtained by cutting out the bottom can be incorporated into the microfluidic device for cell migration assay of the present embodiment. Alternatively, the porous film may be prepared using a technique such as a track etching method.

  The thickness of the porous membrane is preferably 200 μm or less. If the porous membrane is too thick, its significance as a biological tissue model may be diminished. The thickness of the porous membrane is more preferably 100 μm or less, further preferably 50 μm or less, and particularly preferably 20 μm or less. The thickness of the porous membrane has no particular lower limit, but is usually 0.5 μm or more, for example, 1 μm or more or 5 μm or more.

  The porous membrane is preferably incorporated in the substrate at an angle of preferably 45 ° to 90 °, more preferably 70 ° to 90 ° with respect to the horizontal plane. If each flow path in the substrate evolves in a substantially horizontal plane direction and the porous membrane is thus substantially vertically incorporated into the substrate and separates the flow path, the microscope lens is directed in the vertical direction, that is, the thickness direction of the substrate. This makes it possible to observe cell migration permeating through the porous membrane in real time (see FIG. 4). As described later, the incorporation of the film at such an angle can be achieved by forming a cut in the substrate at the above-mentioned angle with respect to the horizontal plane, and inserting the film therein.

  If a device incorporates multiple porous membranes, each of which may have the materials, average pore sizes, thicknesses, and / or angles described above, each of which may be the same as each other between the porous membranes. And may be different. It is preferable that the porous membrane in contact with the first channel has the above-mentioned material, average pore diameter, thickness, and / or angle.

  Methods of forming a microchannel pattern inside a silicone rubber substrate are known to those skilled in the art. For example, a flow channel mold made of an appropriate material is combined with a mold if necessary, a silicone prepolymer is poured into the mold, and the silicone is cured according to the shape of the mold, so that the surface is concave. A substrate having a molded microchannel pattern can be obtained. By joining the flow path side surface of this substrate material to the flat surface of another substrate material and joining, it is possible to obtain a substrate having a flow path space inside the substrate instead of the substrate surface. This bonding can be preferably performed by heating at about 100 ° C. for about 30 minutes, for example, but the bonding method is not limited to this. The material of the mold can be appropriately selected by a person skilled in the art based on ordinary knowledge, but acrylic is suitable for precisely processing a micrometer-scale flow path by laser processing technology combined with drawing software. Resins are particularly preferred. By penetrating the holes from the substrate surface to the respective flow paths, it is possible to form a port capable of filling the flow paths and generating a liquid flow in the flow paths.

  The incorporation of the porous membrane separating the flow channels into the substrate can be achieved, for example, by cutting a suitable portion of the substrate, heating the sandwiched porous membrane to the cut, and joining the cut surfaces. (FIG. 1a). Cutting a silicone rubber substrate is typically accomplished by inserting a sharp blade halfway into the silicone rubber substrate. The notch does not mean that the blade penetrates and the silicone rubber substrate is completely cut into two blocks, but that the substrate block on both sides of the blade is connected by leaving the uncut part without the blade penetrating Means that. A razor is suitable, but not limited to, a cutting means that can be used for this purpose.

  After making the cut, the substrate is subjected to plasma treatment (or corona discharge treatment) while holding the substrate in a bent state so that the cut opening is opened (FIG. 1A, right), and then the porous film is sandwiched and pressed. The present inventor has also found that, when a cut-in surface is joined to produce a membrane-embedded device, the number of individual devices that cause liquid leakage decreases and the yield increases. Since the joining of the cut surfaces by the above-mentioned heating is essentially the same in that the contacted siloxane skeletons are cross-linked, it is not always necessary to reduce the liquid leakage by performing the cross-linking by plasma treatment instead of heating. I could not predict. Further, according to the manufacturing method using the plasma treatment, a porous membrane containing a component that may be denatured or destroyed by heat, such as a scaffold molecule-coated porous membrane, can also be incorporated into the membrane-embedded microfluidic device. Become like As a result, the scaffold molecule can be localized only on the membrane surface, not on the entire inner surface of the flow channel, and the degree of freedom in device design for cell migration assay is greatly improved.

  In order to perform the plasma treatment, the cut substrate is held in a bent state so that the cut opening is opened. In this state, for example, two protrusions (for example, acrylic rods) fixed at a shorter interval than the entire length of the substrate are prepared, one end of the substrate is fixed to one protrusion, and the other end of the substrate is fixed to the other protrusion. This can be achieved by holding the substrate by warping the substrate so that the cut side is a convex surface. The substrate is subjected to plasma processing in this state. After the plasma treatment, it is not necessary to include additional silicone or its prepolymer in the cut when pressing the cut surface with the porous membrane sandwiched between the cuts. Rather, it is preferable to perform pressure bonding without adding silicone or its prepolymer into the cut. It is preferable that only the porous membrane is included in the cut during the pressure bonding.

  The porous membrane preferably has an area smaller than the cross-sectional area of the cut. It is preferable that the porous membrane has a size that does not protrude from the silicone rubber substrate when sandwiched between the cuts. It is preferable that the porous membrane is incorporated into the device in a state where the outer edge is not exposed to the space in the flow channel. That is, it is preferable that the porous membrane is sandwiched between the cuts so that only the membrane surface of the porous membrane is exposed to the space in the flow path, and the membrane edge is not exposed to the space in the flow path.

  When the microfluidic device for cell migration assay of the present embodiment is used, it is preferable to configure a cell migration assay system by including cells in the first channel and a chemoattractant in the second channel ( FIG. 4: In this figure, the “upper channel” is the first channel, the “lower channel” is the second channel, and the “middle channel” is the third channel described later. In this cell migration assay system, it is possible to test the cell migration in which the cells put in the first channel permeate the porous membrane. The cell migration tested here is cell migration dependent on the presence of a chemoattractant placed in the second channel. The test herein may include observing the movement of cells with a microscope (observation may include imaging), and / or measuring the number of cells that have permeated the porous membrane. The cell is a cell having migration or a cell to be tested for the presence or absence of migration. Hereinafter, these are collectively referred to as migratory cells. Migratory cells can be animal cells, plant cells, bacterial cells, fungal cells, and the like, are preferably mammalian cells, and more preferably human cells. Mammalian or human cells can be, for example, immune system cells such as neutrophils and macrophages, or cancer cells.

  At least one of the plurality of channels may further include cells other than the migratory cells. For example, by pre-culturing vascular endothelial cells in the first channel, the porous membrane can be lined with an endothelial cell layer, and cell migration in an in vivo environment can be reproduced more faithfully. (FIG. 4). In another example, the second flow path can include cells that secrete chemoattractants.

  Cells used in this embodiment, especially migratory cells, may be labeled to facilitate observation and / or measurement. Means for labeling cells are known to those of skill in the art, and include, for example, fluorescent labels and radioactive labels.

  The porous membrane (surface and in the pores) and / or the inner surface of the flow channel may be coated with a biomolecule or scaffold molecule such as fibronectin or collagen or a bio-derived composition such as Matrigel. Coating can usually be accomplished by filling the flow path with a solution of these molecules or compositions and then draining excess liquid. Alternatively, as described above, a porous membrane previously coated with these molecules or compositions may be incorporated into the substrate.

  The type of chemoattractant in the present embodiment is to be determined by the user according to each application, and may be any chemoattractant known to those skilled in the art. Examples include formyl peptides and chemokines. One example of a suitable formyl peptide is fMLP (N-formyl-Met-Leu-Phe).

  It is preferable to further provide a third flow path between the first flow path and the second flow path. In this case, the first flow path and the third flow path can be separated by the first porous film, and the second flow path and the third flow path can be separated by the second porous film. Preferably, the first flow path and the second flow path are not adjacent. The first flow path and the second flow path may be separated from each other by, for example, at least 1 mm or more, 2 mm or more, 5 mm or more, or 10 mm or more. As an example, the membrane permeation due to the migration of neutrophil-like cells to the fMLP described above could be observed only slightly in the absence of the third channel, whereas the membrane permeation could be achieved by providing the third channel. Was found to be particularly effective. The reason is that when the third flow path is not provided, the chemoattractant diffuses into the first flow path in a short time and only a small concentration gradient is formed. The attractant diffuses into the first flow path through the third flow path, the concentration gradient of the chemoattractant is formed stably, and many cells migrate and permeate the first porous membrane. There is a possibility. Therefore, it is preferable that the chemoattractant is not introduced into the third flow path, or even if it is introduced, it is introduced at a lower concentration than the second flow path. The term “introduction” here refers not to intrusion through a porous membrane but to artificial introduction through a port or the like.

  The microfluidic device for cell migration assay may be provided with four or more flow paths, and different flow paths may be separated by a porous membrane or separated by other separation means. For example, the third flow path described above may be further divided into a plurality of flow paths.

  In another aspect of the present disclosure, the microfluidic device for cell migration assay described above is used to test cell migration in which cells introduced into the first channel permeate at least one of the porous membranes. A cell migration assay method is provided. Here, the cell migration to be tested is a cell migration dependent on the presence of a chemoattractant introduced into the second channel. The test herein may include observing the movement of cells with a microscope (observation may include imaging), and / or measuring the number of cells that have permeated the porous membrane. When the device has a plurality of porous membranes, it is particularly preferable to test cell migration permeating through the porous membrane in contact with the first flow path, but other porous membranes can be tested.

  Hereinafter, embodiments of the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

[Production of microfluidic device for cell migration assay]
In the microfluidic device for cell migration assay, a template is first prepared, and then the substrate material having a flow path (upper substrate material) is prepared using the template, and the substrate material having no flow path (lower base material) is prepared. Plate material) was also prepared, and the upper and lower substrate materials were directly bonded to each other to obtain a substrate having a flow path therein, and the substrate was obtained by incorporating a porous film into the substrate. Hereinafter, each step will be described in more detail.

Fabrication of a mold A channel pattern and a rectangular pattern of 26 mm × 76 mm were designed using Adobe Illustrator CC software. According to the designed pattern, an acrylic plate (Clarex, Nitto Jushi Kogyo) having a thickness of 1 mm was cut with a laser processing machine (HAJIME, O Laser). See FIG. 1 for the shape of the channel.

  The acrylic plate cut into a rectangle was used as a mold for a lower substrate material having no flow path.

  On the other hand, a syringe (SS-01T, TERUMO) to which a blunt needle (02-165, NIPRO) is attached is filled with an acrylic resin adhesive (Acrysandy), and the same rectangular acrylic as described above is applied by applying this adhesive. An acrylic plate cut in the shape of a channel was adhered onto the plate. This was placed in a circulating thermostatic dryer (MOV-102F, Sanyo Electric) and baked at 65 ° C for 30 minutes. The plywood thus obtained was used as a mold for an upper substrate material having a flow path.

Preparation of Substrate A main component of polydimethylsiloxane (PDMS) (SILPOT 184, Toray Dow Corning) and a curing agent are put in a balance tray (1-5233-04, As One) at a weight ratio of 10: 1 and mixed well. And This was put in a vacuum desiccator (VS, As One) and degassed until no bubbles were found.

  During degassing, a framework was created for pouring the PDMS prepolymer. A Bencott nonwoven fabric (Super CN, Ozu Sangyo) was laid on an acrylic plate, and the mold described above was placed thereon. A mold having a thickness of 3 mm was placed on the mold of the upper substrate material, and a mold having a thickness of 4 mm was placed on the mold of the lower substrate material. The overlap of the acrylic plate, the bencot, the mold, and the formwork was fixed at six places with clips to complete the framework.

  Next, about 4.8 g of the PDMS prepolymer after the completion of degassing was poured into the mold for the frame for the upper substrate, and about 6.4 g for the frame for the lower substrate. This was placed in a vacuum desiccator and degassed again until there were no bubbles. After the deaeration was completed, the substrate was placed in a circulating thermostatic drier and baked at 65 ° C. for 1 hour to cure the PDMS substrate material. After the baking, the substrate material was removed from the mold using tweezers and placed on a slide glass (S1226, MATSUNAMI). Thereafter, each substrate material was cut into a size of 18 mm × 23 mm using a cutter. Further, using a biopsy trepan (BPP-15F, Kai Industries) of φ1.5 mm, holes were made to penetrate from the surface of the upper substrate material having the flow path toward the flow path to form six ports (ports). (See FIG. 1 for the position of.).

  As described above, the upper and lower substrate materials having the same dimensions of 18 mm × 23 mm are directly bonded to each other such that the flow path side of the upper substrate material faces the flat surface of the lower substrate material, and is then subjected to a tabletop blow drying. The substrates were put into a machine (MD-100, Yonezawa) and baked at 100 ° C. for 30 minutes to join the substrate materials to obtain a PDMS substrate having a flow path therein.

Using a scalpel, a PET porous membrane having a pore size of 8.0 μm was cut out from the bottom of the cell culture insert (353093, Falcon) using a scalpel, and cut into 3 mm × 7 mm pieces with scissors. Using a razor, cuts having a depth of about 5 mm were made in two places on the PDMS substrate (see FIG. 1a for cut positions). Thereafter, the cut was pushed open to sandwich the porous membrane, and the cut was closed again. The substrate having the porous film incorporated therein was baked at 100 ° C. for 1 hour in a tabletop blower dryer to join the cut surfaces to complete the device.

  The device in this example has an upper flow path, a middle flow path separated from the upper flow path by the first porous membrane, and a lower flow path separated from the middle flow path by the second porous film. FIG. 1a shows an outline of a step of making a cut in a PDMS substrate and a step of sandwiching a porous membrane in the cut in the process of manufacturing the microfluidic device for cell migration assay of the present embodiment. FIG. 1b is a photograph of the completed device taken from above. In the photograph of FIG. 1b, the upper trapezoidal portion is the upper channel, the centrally long portion is the middle channel, and the lower trapezoidal portion is the lower channel. These channels are visible through the surface of the transparent silicone rubber substrate material. The six structures that look round are ports.

[Culture of HL-60 cells]
The culture of the passage HL-60 cells was performed at 37 ° C. and 5% CO ₂ . As a medium, 500 mL of RPMI1640 (30264-56, Nacalai Tesque), 100 mL of fetal bovine serum (Biowest, inactivated by treatment at 56 ° C. for 30 minutes), 5 mL of 100 mM sodium pyruvate (Nacalai Tesque), and a mixed solution of penicillin-streptomycin (Nacalai Tesque) and 5 mL of 2-mercaptoethanol (Invitrogen) were used.

The cells in the flask cultured for about 5 days were subjected to phase contrast observation with an inverted microscope (CKX53, OLYMPUS) to confirm proliferation. The cell suspension in the flask was transferred to a 15 mL centrifuge tube (1332-015S, WATSON) with a pipette, and centrifuged with a centrifuge (2800, Kubota Corporation) to precipitate the cells (23 ° C., 1200 rpm, 5 minutes) ). After centrifugation, the supernatant was aspirated with an aspirator. After tapping the bottom of the centrifuge tube to loosen the cells, about 2 mL of medium was added and suspended by pipetting. 10 μL of this suspension was placed in a microtube, and 30 μL of trypan blue staining solution (207-17081, Wako Pure Chemical Industries) was added and mixed. Thereafter, the number of cells was measured using a hook Rosenthal hemocytometer. The cell concentration was calculated from the measurement results, and adjusted to a concentration of 5.0 × 10 ⁵ cells / mL by adding a medium. The new flask was further diluted to 5.0 × 10 ⁴ cells / mL by adding 0.5 mL of the adjusted cell suspension and 4.5 mL of medium, and placed in an incubator to resume culture.

Differentiation Induction By adding dimethylsulfoxide (DMSO) to HL-60 cells to induce differentiation into neutrophil-like cells having chemoattractant fMLP, A. Millius and O. Weiner, Methods Mol. Biol., 571, 167 (2009), and the description was also followed in this example. Specifically, first, all the cell suspension in the flask cultured for about 5 days after the passage was transferred to a 15 mL centrifuge tube, and centrifuged with a centrifuge to precipitate the cells (23 ° C., 1200 rpm, 5 rpm). Minutes). On the other hand, in another 15 mL centrifuge tube, 3934 μL of the medium and 66 μL of DMSO (041-29351, Wako Pure Chemical Industries) were added and mixed well to prepare a 1.3% DMSO solution. After the centrifugation, the supernatant was aspirated with an aspirator, the cells were loosened by tapping the bottom of a centrifuge tube, and 1 mL of medium was added and suspended by pipetting. This cell suspension and 4 mL of the above 1.3% DMSO solution were combined, transferred to the original flask, placed in an incubator, and cultured.

[Cell migration test]
Staining solution 10 μL of DMSO was added to Cell Tracker (trademark) Green CMFDA (5-chloromethylfluorescein diacetate) (C7025, Invitrogen), which had been taken out of the frozen storage state and allowed to stand at room temperature for about 30 minutes, and dissolved. The total amount of the solution in the container and 10.789 mL of the serum-free medium were placed in a 15 mL centrifuge tube and mixed by vortexing to obtain a staining solution.

Differentiated cell suspension The cell suspension in the flask 5 to 6 days after the addition of DMSO for differentiation induction was transferred to a 15 mL centrifuge tube and centrifuged by a centrifuge to precipitate cells (23 ° C., 1200 rpm, 5 minutes). The supernatant was aspirated with an aspirator, and about 1 mL of the medium was added thereto, and resuspended by pipetting. 10 μL of this suspension was placed in a microtube and mixed with 30 μL of trypan blue staining solution. The number of cells was measured using a hook Rosenthal hemocytometer. The cell concentration was calculated from the measurement results, and adjusted to a concentration of 5.0 × 10 ⁵ cells / mL by adding a medium.

fMLP solution First, 4.4 mg of fMLP (N-formyl-Met-Leu-Phe) (063-02811, Wako Pure Chemical Industries) was measured with a precision balance. This was placed in a 15 mL centrifuge tube, dissolved in 1 mL of DMSO, and used as a 10 mM stock solution. This was dispensed into microtubes at 50 μL each, and the mixture was frozen and stored in the light-shielded state with aluminum foil.

  10 μL of the above fMLP stock solution and 990 μL of the medium were mixed to prepare a 100 μM fMLP working solution. This solution was stored refrigerated, protected from light with aluminum foil. The working solution was used within one month from the preparation. This working solution was used after being diluted with a medium to a concentration according to the experiment.

Fibronectin solution Fibronectin (354008, Cosmo Bio) removed from the frozen storage state was left at room temperature for about 1 hour. 1 mL of sterilized water was added thereto, and the mixture was left standing for 30 minutes to dissolve (1 mg / mL). This was dispensed into microtubes at 30 μL each and stored frozen.

1x PBS
Five tablets of PBS tablets (T900, TaKaRa) and 500 mL of pure water were placed in a bottle, and dissolved and sterilized in an autoclave.

Implementation of Cell Migration Test The device prepared above was placed in a clean bench the day before the test, and UV-sterilized overnight. The device after UV sterilization was put in a vacuum desiccator and degassed for about 1 hour. The frozen fibronectin solution was placed in a refrigerator and left for about 30 minutes to thaw. 270 μL of 1 × PBS was added to 30 μL of the 1 mg / mL fibronectin solution to prepare a 100 μg / mL fibronectin solution. This fibronectin solution was introduced into all the channels of the degassed device and incubated for 30 minutes. After the incubation, the solution was removed from the channel. In this process, the surface and pores of the porous membrane and the inner surface of the flow channel were coated with fibronectin.

A differentiated cell suspension at a concentration of 5.0 × 10 ⁵ cells / mL, which was fluorescently stained by incubating in a staining solution for 30 minutes, was introduced into the upper channel of the device. At that time, the ports of the middle flow path and the lower flow path were closed with mending tape. Next, the ports of the upper flow path and the middle flow path were closed with a tape, and the fMLP solution was introduced into the lower flow path. Finally, the ports of the upper channel and the lower channel were covered with tape, and the medium was introduced into the middle channel. After introducing the liquids into all the flow paths, a lid was covered with a PDMS substrate having a thickness of 2 mm so as to cover all the ports. The device was placed in a culture chamber (C-070A, blast), and fluorescence microscopy was performed every 15 minutes for 3 hours.

  FIG. 2 shows fluorescence microscope images of the boundary region between the upper channel and the middle channel at 0 hour (upper) and 3 hours (lower) after the start of the culture as described above. When fMLP was not introduced into the lower flow path (0 nM), the fluorescence signal, ie, the presence of neutrophil-like cells, was not substantially detected in the middle flow path even after 3 hours from the culture (FIG. 2, lower left). . That is, neutrophil-like cells did not migrate through the porous membrane in the absence of the chemoattractant. In contrast, when 100 nM fMLP was introduced into the lower channel, a large number of fluorescently labeled neutrophil-like cells were detected in the middle channel after 3 hours from the culture (FIG. 2, lower right). This result indicates that the device was able to accurately reproduce the cell migration phenomenon in which neutrophils were attracted by the chemoattractant and penetrated the gap of the blood vessel wall.

  FIG. 3A shows the result of measuring the number of cells (migrating cells) detected in the middle channel in the experiment of FIG. FIG. 3b shows the results of comparing the numbers of migrated cells after 3 hours when different concentrations of fMLP were introduced into the lower channel. FIG. 3c shows the results of measuring the number of migrating cells when 100 nM or 1 μM Wortmannin was introduced into all the channels in addition to 100 nM fMLP in the lower channel. Wortmannin is an inhibitor of PI3 kinase, a molecule involved in cell migration. It can be seen that Wortmannin suppresses cell migration in a dose-dependent manner. This result further demonstrates the utility of the device in studying cell migration.

  In FIG. 3b, when the concentration of fMLP was increased to 1000 nM, the number of migrating cells tended to decrease. If the concentration of the chemoattractant is too high for the entire system, it is possible that the cells will not be able to effectively sense the concentration gradient.

  Even if the upper flow path and the lower flow path are adjacent to each other and separated by a single porous membrane without providing the middle flow path, cells are migrated even when cells are placed in the upper flow path and fMLP is placed in the lower flow path. Was observed, but cell migration was not as efficient as when the middle channel was provided (data not shown). This is because when the middle flow path is not provided, the chemoattractant diffuses from the lower flow path to the upper flow path in a short time and only a small concentration gradient is formed, but by providing the middle flow path, the chemoattractant flows from the lower flow path to the middle flow path. It is thought that the concentration gradient of the chemoattractant was formed stably in the vicinity of the porous membrane separating the upper flow path and the middle flow path through the upper flow path and the intermediate flow path. Since such a concentration gradient cannot be formed by a conventional assay such as a cell culture insert, the present embodiment can more accurately and accurately evaluate cell migration under a stable concentration gradient of a chemoattractant. it is conceivable that.

  Embodiments of the present invention can be used in medical / biological / pharmaceutical research devices, medical diagnostics, and drug development fields.

Claims (6)

  In a silicone rubber substrate having a porous membrane incorporated therein, the substrate has a first channel into which migratory cells are introduced and a second channel into which a chemoattractant is introduced. A microfluidic device for cell migration assay, wherein the second channel is separated from the second channel by at least one porous membrane.   The method of claim 1, wherein at least one of the porous membranes is incorporated into the substrate by being inserted into a notch formed at an angle of 45 ° to 90 ° with respect to a horizontal plane in the substrate. The microfluidic device for cell migration assay as described above.   The microfluidic device for cell migration assay according to claim 1 or 2, wherein the average pore size of at least one of the porous membranes is 3 to 15 µm.   A third flow path disposed between the first flow path and the second flow path, wherein the first flow path and the third flow path are formed by a first porous membrane; The microfluidic device for cell migration assay according to any one of claims 1 to 3, wherein the microfluidic device is separated and the second flow path and the third flow path are separated by a second porous membrane.   The microfluidic device for cell migration assay according to any one of claims 1 to 4, wherein the silicone rubber includes polydimethylsiloxane.   The cell migration test of the microfluidic device for cell migration assay according to any one of claims 1 to 5, wherein cells introduced into the first flow path penetrate at least one of the porous membranes. Wherein the cell migration is cell migration dependent on the presence of a chemoattractant introduced into the second channel.