Microfluid device and three-dimensional microculture method for cell

Abstract

The invention provides a microfluidic device comprising at least one cell culture chamber, the at least one cell culture chamber being connected to at least two openings, the device being configured to supply at least one physiologically active substance from at least one of the openings to the at least one cell culture chamber in such a manner as to form a concentration gradient or concentration gradients in the at least one cell culture chamber when cells and a hydrogel are introduced into the at least one cell culture chamber to culture the cells in a 3D-gel medium.

Claims

1. A microfluidic device consisting of a first part being formed by a mold and a second part that differs from the first part, wherein the first part and the second part are plate members that are adhered together, wherein the first part and second part comprise at least one cell culture chamber, the at least one cell culture chamber being connected to two or more openings, the device being configured to supply at least one physiologically active substance from at least one of the openings to the at least one cell culture chamber in such a manner as to form a concentration gradient or concentration gradients in the at least one cell culture chamber when cells and a hydrogel are introduced into the at least one cell culture chamber to culture the cells in a 3D gel medium, wherein the diameter of each of the two or more openings is larger than the diameter of the at least one cell culture chamber, wherein the height of each of the two or more openings is greater than the height of the at least one cell culture chamber, wherein the at least one cell culture chamber forms a closed space except for the connection to the two or more openings, and wherein a first opening of the two or more openings is for providing a fresh culture solution to the at least one cell culture chamber, and a second opening of the two or more openings is for discharging used culture solution from the at least one cell culture chamber, wherein the first opening is directly connected to the at least one cell culture chamber, and the second opening is connected to the at least one cell culture chamber through a microchannel.

2. The microfluidic device according to claim 1, wherein the hydrogel is formed inside the at least one cell culture chamber.

3. The microfluidic device according to claim 1, being configured for use in a high-throughput fashion and comprising from 10 to 400 cell culture chambers.

4. A 3D cell microculture method comprising the steps of introducing cells and a fluidized hydrogel into the at least one cell culture chamber of the microfluidic device according to claim 1; converting the hydrogel into a gel; and supplying at least one physiologically active substance from the at least one of the openings in such a manner as to form a concentration gradient or concentration gradients inside the at least one cell culture chamber to culture the cells in the presence of the at least one physiologically active substance.

5. The 3D cell microculture method according to claim 4, wherein the cells are pluripotent stem cells.

6. The 3D cell microculture method according to claim 4, wherein the cells are human pluripotent stem cells.

7. The 3D microculture method according to claim 4, wherein multiple physiologically active substances are supplied into the at least one cell culture chamber in such a manner as to form concentration gradients.

8. The microfluidic device according to claim 1, wherein a flow in a channel connecting one of the two or more openings to the at least one cell culture chamber is coincident with the direction in which the gradient or gradients are formed and the direction in which the cells are introduced.

9. The microfluidic device according to claim 1, comprising 16, 48, 96, or 384 cell culture chambers.

10. The microfluidic device according to claim 1, wherein the at least one cell culture chamber has a height of about 100 to 1,000 μm, a width of about 100 to 1,000 μm, and a depth of about 1,000 to 10,000 μm, and wherein the microchannel that connects the at least one cell culture chamber to the two or more openings has a length of about 1,000 to 10,000 μm and a diameter of about 100 to 1,000 μm.

11. The microfluidic device according to claim 1, wherein the at least one cell culture chamber has a volume of 100 to 2,000 μL.

12. The microfluidic device according to claim 1, wherein the shape of space for culture in the at least one cell culture chamber is a cylindrical shape, a square tube shape, or an elliptic cylindrical shape.

13. The microfluidic device according to claim 1, wherein the two or more openings are positioned within the microfluidic device so as to correspond to the wells of a conventional 16-well, 48-well, 96-well, or 384-well plate.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1: A conceptual diagram of a 3D microenvironmental culture of human pluripotent stem cells in the present invention. A mixtured liquid of a fluidized hydrogel and human pluripotent stem cells is introduced into a microfluidic device to perform a 3D culture. Physiologically active substances necessary for the culture, such as growth factors, diffuse across the hydrogel, and are thereby supplied to the cells. Factors necessary for differentiation can also be optionally introduced.

(2) FIG. 2: A temperature-sensitive phase transition hydrogel (Mebiol®) used in the present invention. Changing the temperature enables the introduction of cells into a gel and the collection of the cells from the gel.

(3) FIG. 3: An experiment operation procedure of the present invention. 1. Preparation of a microfluidic device (μFD). 2. A typical 2D culture of human pluripotent stem cells. 3. The human pluripotent stem cells are collected by an enzyme treatment or a physical treatment, and mixed with the hydrogel. 4. The mixture solution containing the human pluripotent stem cells and the hydrogel is introduced into the μFD, and the temperature is changed to solidify the hydrogel. 5. 3D culture. The medium is replaced as necessary.

(4) FIG. 4: The steps for preparing a microfluidic device. A desired design was drawn by using 3D-CAD, and a mold of the structure was printed by a 3D printer. It is also possible to prepare a mold by typical photolithography or by using an injection-molding mold for mass production. A PDMS liquid material (a mixture of a base and a curing agent) was poured into the mold to prepare a device made of PDMS with a μFD structure.

(5) FIG. 5: Examples of the microfluidic device for a 3D culture according to the present invention. Left: there is one large opening for cell introduction and medium replacement, and one small opening as an outlet, with the two large and small openings connected through a cell culture chamber. A medium, growth factors, and the like are introduced from the large opening to thereby form the concentration gradients. Right: two large openings are connected to a cell culture chamber. It is possible to homogeneously culture cells in the cell culture chamber.

(6) FIG. 6: High throughput screening (HTS) microfluidic device (μFD). The 3D culture of the present invention is also applicable to HTS-μFD.

(7) FIG. 7: Liquid feeding with an autoinjector. In 96-well (left figure) and 384-well (right figure) formats, the correspondence between the pipette tips and HTS-μFD was confirmed.

(8) FIG. 8: A photograph of human pluripotent stem cells that were 3D-cultured in a μFD, and the shape of the colonies formed of human pluripotent stem cells cultured by different techniques. When cultured in a μFD or a suspension culture, the human pluripotent stem cells formed spherical cell aggregates. The traditional 2D culture formed a single-layered colony.

(9) FIG. 9: The confirmation of the expression of human pluripotent stem cell markers (OCT4 and NANOG) by immunofluorescent staining. Both markers exhibited high levels of expression in the human pluripotent stem cells cultured in a μFD.

(10) FIG. 10: The confirmation of the expression of human pluripotent stem cell markers (SSEA4 and TRA-1-60) and a differentiation marker (SSEA1) by flow cytometry. A 3D culture was performed by using three gels with different degrees of solidity (Soft-HG, Mid-HG, and Hard-HG). In every environment, the stem cell markers exhibited high levels of expression, and the differentiation marker did not exhibit the expression.

(11) FIG. 11: The confirmation of the expression of human pluripotent stem cell markers (SOX2, NANOG, and POU5F1 (OCT4)) by quantitative PCR. In every 3D culture (HG), the stem cell markers exhibited high levels of expression as in the typical 2D culture (matrigel MG).

(12) FIG. 12: The “solidity” of the 3D culture environment in a μFD can be changed by the gel concentration. FIG. 12 shows the sizes of human pluripotent stem cell spheres in gels with different degrees of solidity: too soft (45 mg mL.sup.−1), soft (61 mg mL.sup.−1), medium (75 mg mL.sup.−1), and hard (91 mg mL.sup.−1). Although the spheres in the too soft hydrogel can grow, the too soft gel cannot retain the cells.

(13) FIG. 13: High throughput screening (HTS) microfluidic device (μFD). As shown in the photograph, screening using a 3D culture is possible. Cell-cycle analysis. Fewer G2/M phase cells were present than those in the cells in a suspension culture.

(14) FIG. 14: The evaluation of cell activity (ATP) using a firefly luciferase. In every condition, substantially the same ATP activity was confirmed with no damage to the cells.

(15) FIG. 15: The measurement of growth factor diffusion in a μFD/hydrogel. The diffusion of bFGF (molecular weight: 17 kDa) and transferrin (molecular weight: 80 kDa) in gels with different degrees of solidity was evaluated. Each factor was fluorescently labeled. Factors with a smaller molecular weight rapidly diffused.

(16) FIG. 16: The diffusion of fluorescently labeled dextran in a μFD+hydrogel (Soft-HG 61 mg mL.sup.−1). Fluorescently labeled dextran of different molecular weight was prepared, and the way the dextran diffuses was observed with a fluorescence microscope. Larger molecules did not easily diffuse in a hydrogel.

(17) FIG. 17: The diffusion of fluorescently labeled dextran in a μFD+hydrogel (Soft-HG 61 mg mL.sup.−1). Fluorescently labeled dextran of different molecular weight was prepared, and the way the dextran diffuses was observed with a fluorescence microscope. While the fluorescence intensity was measured, the diffusion of the molecules was quantified. The diffusion decreased in accordance with the molecular weight.

(18) FIG. 18: The diffusion of fluorescently labeled dextran in a μFD+hydrogel (Soft-HG 61 mg mL.sup.1). Dextran with a small molecular weight (3 to 5 kDa) was able to rapidly diffuse in a soft gel due to the influence of the solidity of the gel (concentration). However, dextran with a large molecular weight (10 kDa or more) was little affected by the solidity of the gel.

(19) FIG. 19: Colony formation efficiency of human pluripotent stem cells depending on the growth factor concentration gradient in a μFD/hydrogel. Colonies were efficiently formed near the inlet in which the concentration of the growth factor was high.

(20) FIG. 20: High throughput screening (HTS) microfluidic device (μFD) and a conceptual diagram of the microfluidic part. Cells mixed with a hydrogel were introduced into the cell culture chambers. It is possible to form a concentration gradient of a cell stimulant by connecting the tank for the medium for cell maintenance and the tank for the cell stimulation solution to each cell culture chamber.

(21) FIG. 21: Drawings of the designs of the cell culture chamber in a microfluidic device. Multiple concentration gradients can be formed in a single cell culture chamber. Various stimuli can be applied depending on the design of the microfluidic device.

DESCRIPTION OF EMBODIMENTS

(22) The cells used in the present invention are animal cells, preferably vertebrate cells, and particularly preferably mammal cells. Examples of mammals include humans, mice, rats, dogs, monkeys, rabbits, goats, cows, horses, pigs, and cats, with humans being preferable. The cells are preferably pluripotent stem cells. Examples of pluripotent stem cells include stem cells, such as ES cells, iPS cells, mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells, and muscle stem cells. The pluripotent stem cells are preferably stem cells that can differentiate into multiple organs and tissues, such as ES cells and iPS cells. These stem cells are thought to require the concentration gradient(s) of one or multiple physiologically active substances in the process of differentiation. The use of the device and high throughput system according to the present invention enables the assay of substances important for the differentiation of stem cells.

(23) Examples of physiologically active substances (stimulants) supplied, while forming a concentration gradient, include ions, such as calcium ion, potassium ion, magnesium ion, sodium ion, and chlorine ion; cytokines, such as hepatocyte growth factor (HGF), fibroblast growth factor (bFGF)/FGF-2, insulin, transferrin, heparin-binding EGF, gastrin, TGF-β, insulin-like growth factor (IGF-1), parathyroid hormone-related protein (PTHrP), growth hormone, prolactin, placental lactogen, glucagon-like peptide-1 (glucagon-like peptide-1), exendin-4, and KGF (keratinocyte growth factor); amino acids (e.g., Ala, Gly, His, Arg, Lys, Asp, Glu, Asn, Gln, Leu, Ile, Val, Phe, Tyr, Trp, Ser, Cys, Met, Pro, Thr, β-alanine, taurine, and ornithine); neurotransmission substances; carbohydrates (e.g., glucose, fructose, maltose, lactose, and sucrose); carboxylic acids (e.g., acetic acid, pyruvic acid, butyric acid, lactic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, oxalic acid, and α-ketoglutaric acid); lipids (e.g., triglyceride, diglyceride, steroid, monoterpene, diterpene, sesquiterpene, phospholipid, and ganglioside), polyamines (e.g., spermidine and spermine); mucopolysaccharides; glucuronic acid; galacturonic acid; and pH adjusters. Examples of pH adjusters include acids, such as hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid; bases, such as sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, and lithium hydrogen carbonate; and buffer solutions, such as phosphoric acid buffer solutions, citric acid buffer solutions, acetic acid buffer solutions, and boric acid buffer solutions.

(24) As the hydrogel material for the 3D culture, a variety of materials can be used that exhibit fluidity when cells are introduced into the chamber, and that can form a gel by a means such as the addition of other substances or heating (e.g., 37° C.) in the chamber.

(25) Examples of hydrogels used in the present invention include chitosan gel, collagen gel, gelatin gel, peptide gel, fibrin gel, starch, pectin, hyaluronic acid, alginic acid, fibronectin, vitronectin, laminin, alginate, and fibroin. These can be used singly, or in a combination of two or more.

(26) In a preferable embodiment, the hydrogel is preferably a material that undergoes phase transition in accordance with the temperature, for example, a gel that is a liquid at 15° C. or less, but becomes gelatinous in cell culture conditions (37° C.). When cells are introduced into the microfluidic device, cell manipulation is performed at low temperatures; when the cells are cultured, the temperature is increased to 37° C. to allow the material to form a gel. When the cells are collected after culture, the device is placed at low temperatures, allowing the gel to become a liquid. This makes it possible to remove the material.

(27) Increasing the concentration of the gel can solidify the gel, whereas decreasing the concentration of the gel forms a soft gel. The correlation between the gel strength and the gel concentration of various hydrogel materials is known, and a person skilled in the art can readily determine the desired concentration.

(28) In a preferable embodiment of the present invention, the gel used in the 3D culture is a temperature-sensitive gel material. Such a gel material is known, and Mebiol®, for example, can be used.

(29) In a preferable embodiment of the present invention, usable gel materials are, for example, those capable of forming a gel when calcium ions, such as sodium alginate, are added. For example, a mixture liquid containing sodium alginate, a cell culture solution, and cells is introduced into the cell culture chamber, and then the chamber is impregnated with the solution of calcium ions introduced from the opening to fill the chamber with a gel. The gel can also be fluidized by removing calcium ions with a chelating agent.

(30) In a preferable embodiment of the present invention, a usable hydrogel is, for example, one that forms a gel when heated at 37° C. for 30 minutes, such as collagen gel of Nitta Gelatin Inc. The collagen gel degrades when a collagenase acts on the gel. Thus, the cells can be taken out. Besides collagen, hydrogels that are degraded by enzyme activity, such as gelatin, hyaluronic acid, peptide, fibrin, and chitosan, can be preferably used.

(31) The present invention can also examine the cell behavior affected by the solidity of the cellular environment. The diffusion of a physiologically active substance in a gel, unlike in a solution, is dramatically changed by the molecular weight of the substance. This phenomenon also occurs in vivo, and the microfluidic device of the present invention can reproduce the conditions in vivo.

(32) The present invention is particularly useful as a 3D culture method for pluripotent stem cells, such as ES cells and iPS cells. The use of this method enables the regulation and analysis of the function of pluripotent stem cells of mammals, including humans (e.g., human ES cells and human iPS cells), which have been impossible by traditional methods.

(33) FIGS. 1 and 3 show schematic diagrams of the 3D culture method using the microfluidic device of the present invention. FIG. 2 shows an example of the temperature-sensitive phase transition gel.

(34) The microfluidic device of the present invention can be obtained, for example, by forming a mold using a 3D printer as shown in FIG. 4, pouring a starting material into the mold, and solidifying the material, for example, by polymerization. Although FIG. 4 shows a microfluidic device made of PDMS, a person skilled in the art can easily produce a microfluidic device made of other materials in accordance with FIG. 4 and a known method.

(35) The microfluidic device has multiple cell culture chambers, and the device is preferably for use in a high-throughput fashion. Thus, a single device can have about 10 to 400 chambers, for example, 16, 48, 96, or 384 chambers.

(36) In the microfluidic device of the present invention, the cell culture chamber is connected to at least two openings from which cells or a culture solution can be supplied to the chamber (FIGS. 5 and 21). In the cell culture chamber, cells are 3D-cultured, the culture solution is replaced, and at least one physiologically active substance is supplied in such a manner as to form a concentration gradient or concentration gradients. One device preferably has multiple cell culture chambers. A single device provided with 96 or 384 cell culture chambers can be used as a high throughput device (FIGS. 4, 6, and 7).

(37) It is preferable to cover the upper part of the microfluidic device with a lid to prevent the culture solution from evaporating.

(38) FIG. 15 shows the diffusion of physiologically active substances in hydrogels. FIG. 15 shows that a physiologically active substance introduced from the opening diffuses across the hydrogel, and that the diffusion is affected by the molecular weight of the physiologically active substance. The diffusion rate is also affected by the gel strength (FIG. 18). FIG. 12 shows that the gel strength can be changed by the concentration of the hydrogel, and FIG. 13 shows that the high throughput screening (HTS) microfluidic device (μFD) of the present invention can conduct a cell-cycle analysis. FIG. 14 shows based on the ATP activity that the cells cultured in a 3D culture of the present invention are not damaged. FIGS. 16 and 17 show that the diffusion rate of a large physiologically active substance is slow.

(39) In a preferable embodiment of the present invention, a microfluidic device with multiple cell culture chambers is formed, and cells are cultured in the chambers. The markers of the cultured pluripotent stem cells are expressed (FIGS. 9 to 11). The action of a physiologically active substance, such as the colony formation efficiency of the pluripotent stem cells, was affected by the concentration gradient (FIG. 19). FIG. 20 shows an embodiment in which the channel between the stimulation solution tank for supplying a physiologically active substance and the cell culture chamber is narrow so that the physiologically active substance is supplied little by little, thereby forming a concentration gradient, and in which the medium tank (the opening) is directly connected to the cell culture chamber so that the medium can be efficiently supplied and replaced. As shown in FIG. 21, one, or two or more openings for supplying a physiologically active substance (stimulant) may be provided, and multiple substances can be supplied to the 3D-cultured cells at any given timing.

(40) The height of the cell culture chamber is about 100 to 1,000 μm, the width is about 100 to 1,000 μm, and the depth is about 1,000 to 10,000 μm. The length of the microchannel that connects the cell culture chamber to the opening is about 1,000 to 10,000 μm, and the diameter of the microchannel is about 100 to 1,000 μm.

(41) The size of the opening is larger than the diameter of the microchannel. Setting the size of the opening close to the diameter of the cell culture chamber is effective for introducing cells into the chamber. When the opening is connected to the cell culture chamber through a microchannel that is narrower than the diameter of the opening and the diameter of the chamber, the diffusion of a physiologically active substance is restricted, making it easy to form a concentration gradient of the physiologically active substance.

(42) The chamber has multiple (typically two) openings. When a fresh culture solution is provided from one opening of the chamber, the old culture solution is discharged from the other opening through the microchannel, replacing the culture solution.

(43) Examples of materials for the microfluidic device include polysiloxane-based polymers, such as polydimethylsiloxane (PDMS), and diphenyl siloxane; polyolefins, such as silicone resin/silicone rubber, natural rubber, synthetic rubber, polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polymethyl acrylate (PMA), polycarbonate, polyethylene, and polypropylene; homopolymers and copolymers, such as polyurethane, polystyrene, fluorinated polymer (e.g., PTFE and PVdF), polyvinyl chloride, polymethyl hydrogen siloxane, copolymers of dimethyl siloxane with methyl hydrogen siloxane; and blends of these materials. Polysiloxane-based polymers are preferable, and PDMS is more preferable. A microfluidic device with high transparency is preferable because of the ease of evaluating the 3D cell culture. The microfluidic device is preferably excellent in gas permeability, for example, for oxygen and carbon dioxide.

(44) The microfluidic device can be produced by forming a desired mold, and pouring any polymer listed above or its starting material into the mold. The method for producing the mold is not particularly limited, but the method using a 3D printer is preferable.

(45) The microfluidic device has multiple cell culture chambers, and the device is preferably for use in a high-throughput fashion. Thus, a single device can have about 10 to 400 chambers, for example, 16, 48, 96, or 384 chambers.

(46) The chamber preferably contains a small amount of a culture solution and a small number of cells. A single chamber can retain, for example, 100 to 2,000 μL of a liquid or gel in its space. A cell culture can be performed by supplying cells and a culture solution to the space.

(47) The number of cells cultured in one single chamber is typically about 5×10.sup.5 to 0.5×10.sup.5/cm.sup.2. The shape of the space for culture in the chamber is not particularly limited as long as 3D culture is possible. Examples include a cylindrical shape, a square tube shape, and an elliptic cylindrical shape.

(48) In a particularly preferable embodiment, the present invention is supported by the following developments (a) to (d).

(49) (a) Development of a Novel Microfluidic Device for a 3D Culture

(50) The “extracellular microenvironment,” which plays an important role in the regulation of cell function in vivo, was difficult to reproduce in a traditional in vitro experimental system. This is because the traditional cell culture dishes can treat only a large space (about mm to cm), and were not experimental systems on a μm scale required for the reproduction of the extracellular microenvironment. However, microfluidic devices have enabled the production of a very small space on a μm scale, taking a step closer to the reproduction of the extracellular microenvironment.

(51) Nonetheless, in the experimental cell system of the previous microfluidic devices, cells are merely cultured on a plane surface of a culture substrate (e.g., glass or plastic) in a microfluidic device, and this is not truly a 3D culture. To realize a real 3D culture, the present invention uses a hydrogel as an extracellular substrate. The hydrogel is a gel that is formed when a polymer contains water. Mixing a culture solution with the polymer enables the culture of cells in the gel. In addition, changing the polymer concentration allows adjustment of the solidity of the gel, making it possible to investigate “the influence of the solidity of the environment exerted on cells,” which was previously difficult to examine. The diffusion of a substance in the gel depends on the molecular weight. It is also possible to evaluate the diffusion of a substance in the gel and the cell response to the diffusion.

(52) (b) Development of a 3D Culture Method for Human ES/iPS Cells Using Phase Transition Hydrogel

(53) In the cell culture method using a hydrogel, it has been extremely difficult to collect cells without causing damage to the cells. Thus, in a preferable embodiment of the present invention, a reversible-phase-transition hydrogel is used to solve this problem. Although being less adhesive to cells, this hydrogel shows no cytotoxicity, and can be used as a support carrier for a 3D culture. The hydrogel forms a gel in a cell culture environment (37° C.), and transforms into a liquid at a low temperature (20° C.) or less. Thus, the hydrogel can be introduced into or removed from the microfluidic device without affecting the cells.

(54) (c) Development of a Technique for Preparing a Mold for a Microfluidic Device Using a 3D Printer

(55) Although the μFD has various advantages in cell biology, it takes time to prepare the mold in the process of producing the device.

(56) The present invention encompasses the use of a 3D printer to produce a mold. 3D printers can print an intricate 3D structure, and 3D printers for household use are now commercially available. The 3D printer with an X-Y resolution of 50 μm and a Z resolution of 15 μm used in the present invention is sufficient for producing the microfluidic device for a cell culture. The material used for the mold is a plastic resin with thermotolerance of 70° C., which is a relatively low temperature. Thus, when the design was transferred to polydimethylsiloxane (PDMS), which is a material for the microfluidic device, the temperature was maintained at 65° C.

(57) (d) Development of a High-Throughput Screening System Combined with a Microfluidic Device

(58) To efficiently identify physiologically active substances (stimulants) suitable for differentiation while maintaining the function of pluripotent stem cells, the present invention developed a high-throughput screening system combined with the microfluidic device. Physiologically active substances can be supplied to a 3D culture in such a manner as to form concentration gradients. The high-throughput screening system using the device of the present invention can determine what physiologically active substances are suitable to be supplied in what concentration gradient for development and differentiation of cells.

EXAMPLES

(59) The following describes the present invention with reference to the Examples in more detail.

Example 1: Method for Producing a Microfluidic Device Using a 3D Printer

(60) (1) Materials

(61) SYLGARD® 184 Silicone Elastomer kit (base, curing agent) (Dow Corning)

(62) Nunc OmniTray (Thermo scientific 165218)

(63) Glass Bottom Dish (Iwaki Glass Co. Ltd.)

(64) 3D Printer AGILISTA (Keyence)

(65) Desiccator

(66) Corona Fit CFG-500 (Shinko Electric & Instrumentation Co., Ltd.) 3D-CAD (AutoCAD, Blender, and others)

(67) (2) Operation Procedure

(68) Preparation of Mold

(69) 1. A mask of a mold design, which will be a mold for the microchannel structure, is prepared by using 3D image graphics software (3D-CAD).

(70) 2. The mold design is converted into an stf file.

(71) 3. The stf file is transferred to the 3D printer and printed.

(72) Preparation of PDMS with a Microchannel Structure

(73) 1. A silicone elastomer base is mixed with a curing agent in a ratio of 10:1 (weight ratio) using a stirrer (PDMS mixture).

(74) 2. The PDMS mixture is poured into the mold printed by the 3D printer.

(75) 3. Degasification is performed with a desiccator for 30 minutes.

(76) 4. Heating is performed in an oven at 65° C. overnight.

(77) 5. PDMS is collected from the mold.

(78) Preparation of a Microfluidic Device (HTS-μFD, HTNS-μFD)

(79) 1. An OmniTray or glass bottom dish is treated with a corona discharge (using, for example, Corona Fit CFG-500).

(80) 2. The PDMS is surface-treated with corona.

(81) 3. The OmniTray or glass bottom dish is adhered to the PDMS.

(82) 4. Heating is performed in an oven at 65° C. overnight.

(83) 5. The heated product is stored in a desiccator until use.

Example 2: A 3D Culture Method for Human Pluripotent Stem Cells Using Mebiol Gel (Phase Transition Gel)

(84) (1) Materials

(85) Mebiol gel, Mebiol Inc., PMW20-1001 (10 mL for dilution)

(86) mTeSR1

(87) Veritas Corporation, ST-05850

(88) Y-27632

(89) Wako Pure Chemical Industries, Ltd.

(90) 250-00513 (5 mg)

(91) TrypLE Express (1×), Phenol Red Life technologies 12605028 (500 mL)

(92) (2) Operation Procedure

(93) Dissolution of Mebiol Gel

(94) The weight of the Mebiol gel was measured in its dry form, and 6 to 10 mL of mTeSR1 was added to 10 mL of the Mebiol gel for dilution. The mixture was allowed to stand at 4° C. overnight to dissolve the mixture. The concentration of the Mebiol gel in this experiment is shown below.

(95) Soft 61 mg/mL

(96) Medium 75 mg/mL

(97) Hard 91 mg/mL

(98) Making 3D Culture of Human Pluripotent Stem Cells

(99) 1. The fluidic device is sterilized with UV irradiation for 15 minutes.

(100) 2. An mTeSR1 medium containing Y-27632 at a final concentration of 10 is prepared.

(101) 3. The cells on MEF or Matrigel are rinsed with D-PBS(−) twice.

(102) 4. TrypLE Express is added thereto, and the mixture is allowed to stand at 37° C. for 3 to 5 minutes.

(103) 5. TrypLE Express is removed by suction.

(104) 6. When cells on MEF are used, the cell layer is rinsed in the medium to remove the torn MEF.

(105) 7. The cells are collected in mTeSR1+Y-27632 medium.

(106) 8. The cell suspension is pipetted to obtain a single cell preparation.

(107) 9. Centrifugation is performed at 1,000 rpm for 3 minutes.

(108) 10. The supernatant is removed by suction.

(109) 11. The cell pellets are suspended again in mTeSR1+Y-27632 medium.

(110) 12. The number of cells is measured.

(111) 13. 4×10.sup.5 cells are dispensed into a new tube.

(112) 14. Centrifugation is performed at 1,000 rpm for 3 minutes.

(113) 15. The supernatant is removed by suction.

(114) (The following operations are performed on ice to prevent the gel from solidifying, and the pipette tips for use are also cooled on ice.)

(115) 16. The cell pellets are suspended in 100 to 200 μL of Mebiol gel (dissolved in mTeSR1) (4×10.sup.5 cells/100 to 200 μL).

(116) 17. At this stage, Y-27632 at a final concentration of 10 μM is added to the Mebiol gel.

(117) 18. The cell-Mebiol gel suspension is introduced into the fluidic device cooled on ice (2 to 4×10.sup.4 cells/10 μL)

(118) 19. The device is allowed to stand at 37° C. for 5 minutes to form a gel.

(119) 20. An mTeSR1+Y-27632 medium is added to the medium supplier on the device.

(120) 21. To prevent drying, sterile distilled water is added to the dish.

(121) 22. The next day, the medium is replaced with an mTeSR1+Y-27632 medium, and the following days, the medium is replaced with an mTeSR1 medium once a day.

(122) Collection of Cells from a Mebiol Gel

(123) 1. The old medium in the medium supplier is removed.

(124) 2. The device is allowed to stand on ice for 5 minutes.

(125) 3. A new medium is added to the fluid to dilute the gel.

(126) 4. The cells are collected from the fluid.