WO2018225329A1 - Récipient de culture cellulaire, dispositif de culture cellulaire et procédé de culture cellulaire - Google Patents

Récipient de culture cellulaire, dispositif de culture cellulaire et procédé de culture cellulaire Download PDF

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WO2018225329A1
WO2018225329A1 PCT/JP2018/010440 JP2018010440W WO2018225329A1 WO 2018225329 A1 WO2018225329 A1 WO 2018225329A1 JP 2018010440 W JP2018010440 W JP 2018010440W WO 2018225329 A1 WO2018225329 A1 WO 2018225329A1
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cell
cell culture
fluid
culture
cells
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Japanese (ja)
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優史 丸山
靖彦 多田
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株式会社日立製作所
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    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a cell culture container.
  • the cell culture container according to the present invention includes a cell support, and the cell support is composed of a fluid material at least a part of the surface in contact with the cells, and the water content of the fluid material is relatively low.
  • the cell culture using the cell culture container of the present embodiment can be handled in the same manner as a conventional solid culture container at the time of cell proliferation, and thus has high convenience and high proliferation. Furthermore, the three-dimensional mass formed by spontaneous assembly of cells has high adhesion to the surface of the cell culture container. By controlling the viscosity and surface modification of the flowable material, the configuration of the cell container can be easily customized according to the cell.
  • FIG. 1 shows a cell culture container provided according to this embodiment.
  • the cell culture vessel 1 has a cell support 2 for adhering cells, and the cell support 2 includes a fluid material 3 having fluidity on the entire surface or a part of a surface in contact with the cell.
  • cells are active in a complex environment involving multiple forces such as surface tension, polymerization / depolymerization of intracellular skeleton, cell-scaffold adhesion, cell-cell adhesion.
  • a solid scaffold such as a polystyrene culture vessel, and many culture conditions are known for obtaining high productivity.
  • fluid scaffold in which cells have fluidity (hereinafter referred to as fluid scaffold) equivalent to a solid scaffold
  • the force that the cells receive from the surface of the fluid scaffold must be equivalent to that of the solid scaffold.
  • the displacement of the scaffold is sufficiently slow relative to the force that the cell acts on the surface, so the force that the cell receives from the surface of the fluid scaffold and the cell from the surface of the solid scaffold It is thought that the power received by is equal.
  • a fluid scaffold can behave in the same manner as a solid scaffold depending on conditions.
  • the force that the cells act on the scaffold surface varies depending on the cell density. In the case of a single cell, there is no interaction between cells, but when the cell density increases and the interaction between cells increases, it tries to start movement in the direction in which the cells aggregate, The acting force increases.
  • the force generated by the cells varies depending on the cell type, phenotype, culture conditions and the like. It is also affected by the adhesion factor of the scaffold surface and the ECM. Therefore, appropriate conditions may vary depending on the subject to be cultured. Appropriate conditions mentioned here are, for example, the viscosity, surface functional group, surface state, density, etc. of the cell support having fluidity. These details will be described later.
  • the mechanobiological stimulation received by the cells differs from the conventional solid scaffold.
  • mechano-biological control may change cytoskeletal metabolism, gene expression patterns, cell migration, etc., and may change cell characteristics .
  • the fluid scaffold material according to the present embodiment can be used not for three-dimensional cell culture but for mechanobiological cell property control. is there.
  • the cell type to which the culture container of this embodiment can be applied is not limited as long as it is an adhesive cell. Moreover, there is no restriction
  • adhesive cells examples include stem cells (mesenchymal stem cells, ES cells, iPS cells, etc.), epithelial cells (vascular epithelial cells, bile duct epithelial cells, etc.), endothelial cells (intravascular non-cells, lymphatic endothelium). Cells, etc.), fibroblasts (NIH3T3, etc.), hepatocytes, pancreatic islet cells, neurons, cardiomyocytes, myoblasts, cancer cells, macrophages, HeLa cells, CHO cells, etc. It is not limited. Moreover, the cell to culture does not need to be 1 type, You may apply to a co-culture system.
  • the floating cells can be cultured while attached to the cell support.
  • the fluidity effect of the scaffold is considered to be smaller than the effect obtained in adherent cells.
  • liquidity will become small in the cell with small interaction between cells.
  • the cell support having fluidity can be realized by using a liquid or semi-solid polymer (hereinafter referred to as fluid material) which is insoluble or hardly soluble in the medium.
  • fluid material a liquid or semi-solid polymer which is insoluble or hardly soluble in the medium.
  • a flowable material that is sufficiently low in cytotoxicity, it does not necessarily need to be insoluble in the medium, but it can affect applications such as cell assays and transplants, so It is desirable that the solubility of is lower. It is also desirable that the flowable material is not emulsified by amphiphilic components that may be included in the medium.
  • hydrogels are difficult to prepare, handle due to problems such as low mechanical strength and weakness to drying.
  • the cell support having fluidity has low mechanical strength, but even if scratches, cracks, defects, etc. occur, it is repaired by the fluidity, so that it is easy to handle.
  • materials with low moisture content are easy to handle with less drying problems. From this point of view, the lower the water content, the less the problem, preferably 20% or less, more preferably 5% or less, and even more preferably 1% or less.
  • the fluid material may be used in bulk or may be used by coating on the surface of some substrate. Further, the surface of the fluid material may be treated by a physical / chemical method. Physical / chemical methods include, for example, light irradiation, plasma treatment, corona discharge treatment, UV / O3 treatment, surfactant treatment, adsorption of chemical substances, adsorption of biomolecules, addition of particulate substances and fibrous substances, Including but not limited to patterning, any combination thereof, and the like.
  • FIG. 2 is a schematic view of a cell culture container obtained by subjecting a fluid material to a predetermined treatment. By the above treatment, a surface component 4 different from the fluid material is generated on the fluid material 3.
  • such a surface can be formed by a method of contacting a solution containing a cell adhesion factor or ECM, contacting serum, or contacting a medium.
  • the flowable material may be a single component or a mixed system of a plurality of components. Moreover, molecular weight distribution is not limited as long as fluidity is ensured.
  • a flowable material is preferable if the transparency is high because cells can be easily observed. However, it is not necessarily transparent.
  • the viscosity of the flowable material needs to satisfy the range that the cells can be extended and that the deformation is easy with respect to the cohesive force between the cells generated at a high cell density.
  • This range depends on the cell and phenotype, but according to our experimental results, it is preferably 1 ⁇ 10 3 mPa ⁇ s to 1 ⁇ 10 9 mPa ⁇ s, more preferably 2 ⁇ 10 3 mPa ⁇ s to 1 ⁇ 10 8 mPa ⁇ s.
  • it is more preferably 5 ⁇ 103 mPa ⁇ s or more and 1 ⁇ 107 mPa ⁇ s or less.
  • the flowable material may be a Newtonian fluid or a non-Newtonian fluid.
  • the movement of cells is generally very slow, and in order to obtain a sufficient effect with a non-Newtonian fluid, precise adjustment of physical properties is required so as to obtain an appropriate viscosity with respect to the movement speed of the cells.
  • the specific gravity of the flowable material may be larger than, equal to, or smaller than that of the medium.
  • the flowable material when the flowable material is coated on the surface of the substrate, it is desirable that the flowable material does not peel from the substrate, so that the affinity between the flowable material and the substrate is sufficiently high, and surface tension and It is necessary to be able to antagonize the force that can deform a fluid material such as buoyancy and gravity.
  • the specific gravity of the flowable material is heavier than that of the medium, it can be easily handled as long as it is used for a portion such as the bottom of the dish.
  • the surface of a substrate having a certain three-dimensional shape is coated and used, sufficient interaction is required between the flowable material and the substrate to prevent the coating from falling.
  • the storage energy and kinetic energy are negligible among the three components of the medium, flowable material, and substrate, and the flowable material is stable when the sum of surface energy and the sum of potential energy exist around the minimum value. It can be expressed that the coated state on the surface of the substrate is maintained. When the coating of the flowable material is unstable, the flowable material deforms and shifts to a more stable state. In this case, the mode (1) in which the contact area between the culture medium and the substrate does not change due to the deformation. , Can be classified into two modes (FIG. 3). In order for the fluid culture vessel to exist stably, it is necessary that neither of these progress.
  • mode (1) is a deformation in which, for example, when the flowable material 3 having a specific gravity smaller than the culture medium 6 is coated on the bottom surface of the dish, the flowable material 3 is deformed and a part of the coating is lifted. . The contact area between the deformed portion 7 of the fluid material and the culture medium 6 is increased.
  • the state where the fluid material is stably coated is a state where the contact area between the fluid material and the medium does not increase spontaneously. That is, in a state where the contact area between the fluid material and the culture medium increases, the total energy change is positive and energy loss occurs.
  • a state can be expressed by Equation 1. Since ⁇ V ⁇ h / ⁇ S is a term that depends on the shape of the deformed portion, and ⁇ , ⁇ , and g are constants, it can be said that this equation substantially describes the shape of the deformation that can proceed spontaneously. Qualitatively, the affinity between the flowable material and the medium is low, and the density difference between the flowable material and the medium is large, suggesting that deformation accompanied by volume change is more likely to proceed over a wide range.
  • mode (2) is a deformation in which, for example, when the flowable material having a specific gravity smaller than the medium is coated on the bottom surface of the dish, the flowable material is deformed and the base material of the coating is exposed. The contact area between the base material 5 and the culture medium 6 is increased.
  • the state where the fluid material is stably coated is a state where the contact area between the culture medium and the substrate does not increase spontaneously. That is, in a state where the contact area between the culture medium and the substrate increases, the change in total energy is positive and energy loss occurs.
  • the potential energy is the same as in Equation 1, but regarding the surface energy, there are changes in the contact area between the fluid material and the medium, the medium and the substrate, and the fluid material and the substrate. Since the increase in the contact area of the substrate is equal to the decrease in the contact area between the flowable material and the substrate, it is simplified as shown in Equation 2.
  • the lower the affinity between the fluid material and the substrate the easier the deformation proceeds.
  • examples of the shape of the fluid culture vessel in which modes (1) and (2) are difficult to proceed include, as an example, a thinly coated fluid material on a substrate having a low affinity for the culture medium.
  • Mode (1) which can be deformed only by potential energy due to the thin coating
  • mode (2) which is deformed by surface energy gain due to increased contact area between the medium and the substrate due to low affinity between the medium and the substrate. This is because it can be suppressed.
  • the metastable state is caused by some cause, if the transition to the stable state is sufficiently slow, it can be used for cell culture. Even when there is a possibility that the stable state is temporarily lost during the culture, the cell can be used for the cell culture if the transition to the stable state is sufficiently slow.
  • a simple method for judging the stability of a flowable material in water is to leave the flowable material in water at 37 ° C. for 24 hours. If the stability is insufficient, the above-described mode (1) or mode (2) deformation is observed. When it is stable or the displacement per hour is sufficiently small, no clear deformation is observed.
  • fluid materials that have low cytotoxicity and are insoluble in the culture medium include polybutene, polyisobutylene, hydrogenated polybutene, hydrogenated polyisobutylene, block copolymers containing these, hydrocarbons such as liquid paraffin, Polymer materials such as siloxanes such as silicone and halogens such as perfluorocarbon and perfluoroether can be used, but are not limited thereto. Further, the terminal functional group, the branched / crosslinked structure, the tacticity, etc. of such a polymer material are not limited as long as it exhibits fluidity.
  • the fluid material a material system in which the viscosity depends on the molecular weight and the mixing ratio of the components is desirable because it is easy to control the fluidity of the cell support, but is not limited thereto.
  • Ionic liquids that are insoluble in the culture medium can also be candidates for flowable materials, but they are not always suitable as cell supports because they need to be ionic species with low cytotoxicity and fluidity is difficult to control. Not.
  • non-linear hydrocarbon materials such as polybutene and polyisobutylene are particularly preferable as the flowable material from the viewpoint of the viscosity range and surface energy.
  • Polybutene and polyisobutylene have very low solubility in water and are almost never mixed into the medium. They can be easily removed even if they are mixed. They are used for food ingredients, cosmetic ingredients, medical adhesives, etc. It is useful because it is a very safe material that can be expected to have little adverse effects even if it is mixed in a small amount of medium.
  • Such a non-linear hydrocarbon-based material generally has a density of less than 1 g / mL.
  • the combination of the fluid material and the substrate is important.
  • polybutene is used as the flowable material
  • low density polyethylene is used as the base material
  • a polybutene layer having a thickness of 30 ⁇ m is formed on the low density polyethylene.
  • the substrate not only polyethylene but also polyalkylene other than polyethylene, such as polypropylene and polybutene copolymer, a material having a surface grafted with polyalkylene, and the like can be used, but not limited thereto.
  • the fluidity of the film may depend on the film thickness, so that the physical properties may be controlled not only by the material composition but also by the film thickness.
  • the film thickness is small, the specific surface area of the surface in contact with the base material or the culture medium with respect to the volume of the fluid material increases, so the influence of buoyancy and gravity becomes relatively large, and the layer of fluid material becomes It becomes easier to exist more stably.
  • the shape of the base material coated with the fluid material is not limited. For example, a plane, an aspect, a film, a mesh, a fiber, a cloth, a particle, a pillar, an uneven surface, and the like can be considered.
  • FIG. 4 the example of the film-like base material 8, the mesh-like base material 9, and the pillar-shaped base material 10 is shown.
  • Examples of the coating method include a method of applying a bulk fluid material, a method of applying a fluid material solution and drying a solvent, and a method of vaporizing a vaporized fluid material. Not.
  • a fluid culture container that can be handled in the same manner as a normal culture dish is obtained by coating a fluid material on a film-like base material and attaching it to the bottom of the culture dish.
  • the cell can be easily moved by peeling the film to which the cell is adhered from the bottom surface.
  • the convenience is high (FIG. 5).
  • the film-like substrate can be attached as it is to the culture dish using the flowable material, so that it can be easily produced.
  • the fluidic material layer is positively fluidized by stretching / compressing the substrate during culture. Can do.
  • mechanical stimulation can be given to the cells during the culture, and dynamic mechanobiological control can be performed.
  • the sterilization method of the flowable material should be selected according to the characteristics of the flowable material.
  • ⁇ rays, ethylene oxide gas, UV irradiation, ethanol, drying heating, saturated steam heating, and the like can be considered.
  • a general method such as introducing a cell suspension into the fluid culture vessel can be used (FIG. 7).
  • proteolytic enzyme can be used to peel off cells adhered to the surface of the fluid material (FIG. 8).
  • the surface of the flowable material is coated with something such as a stimulus-responsive molecule, cell detachment by a stimulus response can also be performed.
  • the invasiveness to the cells is low when the cells are detached by a liquid flow as compared to the case of using a solid scaffold.
  • the following describes a specific example of production on a culture vessel.
  • Example 1 a fluid culture vessel was produced using a film-like substrate using polybutene (hereinafter referred to as polybutene 1) having a viscosity of 2.0 ⁇ 10 5 mPa ⁇ s at 37 ° C. as a fluid material.
  • polybutene 1 polybutene having a viscosity of 2.0 ⁇ 10 5 mPa ⁇ s at 37 ° C.
  • the low-density polyethylene film was immersed in a 33 wt% toluene solution of the polybutene 1 and pulled up, and then air-dried to coat the film-like substrate with a fluid material.
  • the obtained film was affixed to the bottom of the TCPS culture vessel and dried for 1 day under reduced pressure to remove volatile components.
  • the obtained fluid culture vessel was returned to atmospheric pressure, and the surface was annealed by allowing it to stand at room temperature for one day or at 40 ° C. for half a day.
  • the fluid culture vessel is referred to as a culture vessel 1.
  • the culture vessel 1 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed.
  • the water content of polybutene 1 after standing for 24 hours was 0.5% or less.
  • Example 2 a fluid culture vessel was produced with a film-like substrate using polybutene (hereinafter referred to as polybutene 2) having a viscosity of 7.5 ⁇ 10 4 mPa ⁇ s at 37 ° C. as a fluid material.
  • polybutene 2 having a viscosity of 7.5 ⁇ 10 4 mPa ⁇ s at 37 ° C.
  • a low-density polyethylene film was immersed in a 33 wt% toluene solution of the polybutene 2 and pulled up, and then air-dried to coat a fluid material on the film-like substrate.
  • the obtained film was affixed to the bottom of the TCPS culture vessel and dried for 1 day under reduced pressure to remove volatile components.
  • the obtained fluid culture vessel was returned to atmospheric pressure, and the surface was annealed by allowing it to stand at room temperature for one day or at 40 ° C. for half a day.
  • the fluid culture vessel is referred to as a culture vessel 2.
  • the culture vessel 2 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed.
  • the water content of polybutene 2 after standing for 24 hours was 0.5% or less.
  • Example 3 a fluid culture vessel was manufactured using a mesh-like substrate using polybutene 1 as a fluid material.
  • a polypropylene mesh was immersed in a 10 wt% toluene solution of polybutene 1, pulled up, and then air-dried to coat a fluid material on the mesh substrate.
  • the obtained mesh was placed on the bottom surface of the low adhesion culture vessel and dried under reduced pressure for 1 day to remove volatile components.
  • the obtained fluid culture vessel was returned to atmospheric pressure, and the surface was annealed by allowing it to stand at room temperature for one day or at 40 ° C. for half a day.
  • the fluid culture vessel is referred to as a culture vessel 3.
  • the culture vessel 3 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed.
  • the water content of polybutene 1 after standing for 24 hours was 0.5% or less.
  • Example 4 a fluid culture container was produced with a film-like substrate using polybutene 2 as a fluid material.
  • a low-density polyethylene film was immersed in a 33 wt% hexane solution of polybutene 2, pulled up, and then air-dried to coat a fluid material on the film-like substrate.
  • the obtained film was affixed to the bottom of the TCPS culture vessel and dried for 1 day under reduced pressure to remove volatile components.
  • the obtained fluid culture vessel was returned to atmospheric pressure, and the surface was annealed by allowing it to stand at room temperature for one day or at 40 ° C. for half a day.
  • the fluid culture vessel is referred to as a culture vessel 4.
  • the culture vessel 4 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed.
  • the water content of polybutene 2 after standing for 24 hours was 0.5% or less.
  • Example 5 a fluid culture vessel was produced with a film-like substrate using a mixture of polybutene 1 and polybutene 2 as a fluid material.
  • a low-density polyethylene film was immersed in a 33 wt% toluene solution of a 1: 1 mixture of polybutene 1 and polybutene 2, pulled up, and then air-dried to coat a fluid material on the film-like substrate.
  • the obtained film was affixed to the bottom of the TCPS culture vessel and dried for 1 day under reduced pressure to remove volatile components.
  • the obtained fluid culture vessel was returned to atmospheric pressure, and the surface was annealed by allowing it to stand at room temperature for one day or at 40 ° C. for half a day.
  • the fluid culture vessel is referred to as a culture vessel 5.
  • the culture vessel 5 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed.
  • the water content of the mixture of polybutene 1 and polybutene 2 after standing for 24 hours was 0.5% or less.
  • Example 6 a fluid culture vessel was produced on Teflon (registered trademark) using polybutene 1 as a fluid material.
  • a 33 wt% toluene solution of polybutene 1 was coated on a Teflon (registered trademark) container having fine irregularities on the surface, air-dried, and dried under reduced pressure for 1 day to remove volatile components.
  • the obtained fluid culture vessel was returned to atmospheric pressure, and the surface was annealed by allowing it to stand at room temperature for one day or at 40 ° C. for half a day.
  • the fluid culture vessel is referred to as a culture vessel 6.
  • the culture vessel 6 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed.
  • the water content of polybutene 1 after standing for 24 hours was 0.5% or less.
  • Example 7 the surface modification of the culture vessel 1 by molecular adsorption was examined.
  • a sodium stearate aqueous solution, a collagen aqueous solution, and a fibronectin aqueous solution were respectively added and incubated for 30 minutes. Thereafter, the solution was removed, washed with pure water, and dried. Surface analysis confirmed the presence of the acted molecules. An increase in wettability was also confirmed.
  • Example 8 the surface modification of the culture vessel 1 by adsorption of particles or the like was examined.
  • each of a dispersion solution of crosslinked agarose beads, polystyrene beads, and cellulose nanofibers was placed and incubated for 30 minutes. Thereafter, the solution was removed, washed with pure water, and dried. Surface analysis confirmed the presence of particles that were acted on.
  • Example 9 a flowable culture vessel coated with a polybutene-polyacrylic acid block copolymer having a water content of 4% as a flowable material was produced.
  • a 10 wt% THF solution of polybutene-polyacrylic acid block copolymer is coated on a Teflon (registered trademark) container with fine irregularities on the surface, air-dried, and then dried for one day under reduced pressure to remove volatile components. did.
  • the obtained fluid culture vessel was returned to atmospheric pressure, and the surface was annealed by allowing it to stand at room temperature for one day or at 40 ° C. for half a day.
  • the fluid culture vessel is referred to as a culture vessel 9.
  • the culture vessel 9 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed.
  • the water content of polybutene 1 after standing for 24 hours remained at 4% or less.
  • Comparative Example 1 a fluid culture vessel was produced using a film-like substrate in the same manner as in Example 1, using Polybutene 3 having a viscosity of 7.0 ⁇ 10 2 mPa ⁇ s at 37 ° C. as the fluid material.
  • the fluid culture container is referred to as a comparative container 1.
  • Comparative container 1 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed.
  • the water content of polybutene 3 after standing for 24 hours was 0.5% or less.
  • Comparative example 2 a fluid culture vessel was produced on glass using polybutene 2 as a fluid material.
  • a 33 wt% toluene solution of polybutene 2 was coated on a glass petri dish, air-dried, and then dried for one day under reduced pressure to remove volatile components.
  • the obtained fluid culture vessel was returned to atmospheric pressure, and the surface was annealed by allowing it to stand at room temperature for one day or at 40 ° C. for half a day.
  • the fluid culture container is referred to as a comparative container 2.
  • Comparative Example 3 a fluid culture vessel was produced using a film-like substrate in the same manner as in Example 4 using polybutene 4 having a viscosity of 1 ⁇ 10 10 mPa ⁇ s at 37 ° C. as the fluid material.
  • the fluid culture container is referred to as a comparative container 3.
  • Comparative container 3 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed.
  • the water content of polybutene 4 after standing for 24 hours was 0.5% or less.
  • Comparative example 4 a fluid culture vessel was produced using a film-like substrate in the same manner as in Example 4 using silicone oil 5 having a viscosity of 6 ⁇ 10 2 mPa ⁇ s at 37 ° C. as the fluid material.
  • the fluid culture container is referred to as a comparative container 4.
  • the coating peeled off.
  • the water content of the silicone oil 5 after standing for 24 hours was 1% or less.
  • Comparative Example 5 a fluid culture vessel of TCPS was manufactured using acrylamide hydrogel having a viscosity of 2 ⁇ 10 5 mPa ⁇ s at 37 ° C. and a water content of 80% as a fluid material.
  • the fluid culture container is referred to as a comparative container 5.
  • Comparative container 5 was allowed to stand at 37 ° C. in water for 24 hours, but the coating was stable and no peeling or the like was observed. The moisture content of the acrylamide hydrogel after standing for 24 hours remained at 80%.
  • Example 10 cell culture was performed using the culture vessels 1 to 5 and 9. Prior to cell culture, each culture container was incubated for 30 minutes with a medium to be used or a solution of an adhesion factor suitable for the cells to be cultured, thereby improving surface cell adhesion. The culture medium and seeding conditions used were recommended for the cell type to be cultured. As a result, in all cases, the behavior of the cell spreading in the early stage and the spontaneous aggregation of the cell in the later stage was observed, and the fluidity effect was recognized (FIG. 6). As shown in FIG. 6, the effect of viscosity on cell morphology is large.
  • Comparative Example 6 cell culture was performed in the same manner as in Example 10 using Comparative Containers 1, 3, 5, and TCPS. As a result, in the comparative container 1 in which the fluidity is too high, the cell extension was not observed, but in the comparative container 3 having a too low fluidity or the solid TCPS, the cells were extended, but the monolayer state was maintained, and It was confirmed that spontaneous aggregation of cells does not proceed unless the fluidity is within a range. Moreover, in the comparative container 5 having a high water content, cells infiltrated into the fluid material, and spontaneous aggregation due to fluidity was not observed.
  • Examples and comparative examples> In the case of using a base material to hold the flowable material, in order to function as a culture vessel, the viscosity is only within a predetermined range (1 ⁇ 103 mPa ⁇ s or more and 1 ⁇ 109 mPa ⁇ s or less at 37 ° C.). Instead, it is necessary that the flowable material does not peel from the substrate. As described in Comparative Example 2, even when the polybutene 2 having a viscosity in the above-described predetermined range is used, when the substrate is glass, the coated state is not near the minimum value in terms of energy in water, and is peeled off. Is energetically advantageous and the coating peels off.
  • Comparative Example 2 is a combination in which the flowable material is peeled off, and the other cases are NG because the viscosity and moisture content are outside the above-described predetermined ranges.
  • SYMBOLS 1 ... Cell culture container, 2 ... Cell support body, 3 ... Fluid material, 4 ... Surface component 5 ... Base material, 6 ... Medium, 7 ... Deformation site

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Abstract

La présente invention permet d'améliorer la commodité, la capacité de prolifération, l'adhésivité et l'aptitude à la personnalisation d'une culture cellulaire tridimensionnelle. Le récipient pour culture cellulaire selon la présente invention est pourvu d'un élément de support de cellules, au moins une partie d'une surface de l'élément de support de cellule, ladite surface devant être en contact avec les cellules, étant formée d'un matériau fluide, et le matériau fluide présentant une teneur en eau relativement faible.
PCT/JP2018/010440 2017-06-07 2018-03-16 Récipient de culture cellulaire, dispositif de culture cellulaire et procédé de culture cellulaire WO2018225329A1 (fr)

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JP2017112203A JP2018201440A (ja) 2017-06-07 2017-06-07 細胞培養容器、細胞培養装置及び細胞培養方法
JP2017-112203 2017-06-07

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001190267A (ja) * 2000-01-07 2001-07-17 Mitsui Chemicals Inc ポリオレフィン樹脂組成物からなる培養容器
JP2013099285A (ja) * 2011-11-08 2013-05-23 Dainippon Printing Co Ltd 細胞培養容器の製造方法
WO2016043488A1 (fr) * 2014-09-19 2016-03-24 (주)세포바이오 Méthode permettant d'induire une différenciation ostéogénique tridimensionnelle de cellules souches au moyen d'hydrogel
JP2016111988A (ja) * 2014-12-17 2016-06-23 株式会社リコー 三次元細胞集合体、並びにその製造方法及び形成用溶液

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001190267A (ja) * 2000-01-07 2001-07-17 Mitsui Chemicals Inc ポリオレフィン樹脂組成物からなる培養容器
JP2013099285A (ja) * 2011-11-08 2013-05-23 Dainippon Printing Co Ltd 細胞培養容器の製造方法
WO2016043488A1 (fr) * 2014-09-19 2016-03-24 (주)세포바이오 Méthode permettant d'induire une différenciation ostéogénique tridimensionnelle de cellules souches au moyen d'hydrogel
JP2016111988A (ja) * 2014-12-17 2016-06-23 株式会社リコー 三次元細胞集合体、並びにその製造方法及び形成用溶液

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