JP4033265B2 - Cell tissue microdevice - Google Patents

Description

  The present invention relates to a microdevice for forming a cellular tissue body.

  Currently, the development of medical technology using cell culture technology and the development of pharmaceuticals are actively performed. That is, for example, a technique for culturing cells that express a function peculiar to a tissue or organ of a living body uses the cell as an in vitro model of the tissue or organ, and evaluates a drug toxicity test or an endocrine disrupting action. It is being applied to screening for new drugs.

  In such a cell culture technique, in general, cells are cultured in a single layer (that is, two-dimensionally) on a flat base material coated with a cell adhesive substance such as collagen. When the primary hepatocytes and the like taken out from the above are cultured in such a monolayer state, the functions peculiar to the liver disappear or die within a very short period of time.

  In contrast, in recent years, instead of culturing primary hepatocytes in a monolayer, cells can be cultured as a cell tissue that is an aggregate of three-dimensionally linked cells. It has been found that can be maintained over a longer period of time.

  As a method for forming such a hepatocyte tissue body, conventionally, for example, in Patent Document 1, a specific growth factor is added to the culture solution to form a spherical hepatocyte tissue body in the pores of the polyurethane foam. Is disclosed.

Further, in Patent Document 2, a hepatocyte tissue body is formed on a plane coated with a polymer substance composed of a monomer having phenylboronic acid, a monomer having an amino group, and a 2-hydroxyethyl methacrylate copolymer. Is disclosed.
Japanese Patent Laid-Open No. 10-29951 Japanese Patent No. 3020930

  However, in the above conventional method, a culture substrate surface to which cells adhere relatively weakly (that is, cell adhesion is relatively low) is used, and the formed cell tissue is detached from the culture substrate surface. In order to be in a floating state in the culture solution, for example, in the process of culturing, when the culture solution that has consumed nutrients is removed and replaced with a fresh culture solution, cells and In some cases, cell tissues were removed, making it impossible to continue culturing thereafter.

  In addition, in the above conventional method, since a relatively long period is required until a cell tissue body is formed from cells, for example, after being removed from the living liver, the function peculiar to the liver is deteriorated over time. When primary hepatocytes or the like are used, the function of the formed hepatocyte tissue structure may be insufficient.

  The present invention has been made in view of the above problems, and provides a microdevice capable of forming a cell tissue in a short period of time and culturing the formed cell tissue for a long period of time. Is one of its purposes.

  In order to solve the above-described conventional problems, a cell tissue microdevice according to an embodiment of the present invention includes a cell holding chamber for holding cells, and the cell holding chamber includes at least one tissue forming region. Including a bottom surface, an inflow port for allowing the culture solution to flow into the cell holding chamber, and an outflow port for allowing the culture solution to flow out of the cell holding chamber. It is characterized by including one 1st area | region which shows adhesiveness, and the 2nd area | region which surrounds the said 1st area | region and shows low cell adhesiveness compared with the said 1st area | region.

  In addition, the bottom surface of the cell holding chamber may include a third region that surrounds the tissue formation region and exhibits lower cell adhesion than the second region.

  Further, the cell holding chamber bottom surface includes a plurality of the tissue body forming regions, and includes a third region that separates the plurality of tissue body forming regions from each other and exhibits low cell adhesion compared to the second region. It is good as well.

  The cell holding chamber bottom surface may include a cell holding cavity, and the cell holding cavity bottom surface may include one tissue body forming region.

  Moreover, the diameter of the tissue body formation region may be in the range of 2 to 50 times the diameter of the cells.

  The first region may be formed near the center of the tissue formation region.

  In the first region, a cell adhesive substance or a derivative thereof obtained or synthesized from a living body may be immobilized.

  Further, a cell tissue body may be held in the first region.

  The cell tissue body forming method according to an embodiment of the present invention includes a step of seeding cells in the cell holding chamber of the cell tissue microdevice, and the cell holding in which the cells are seeded from the inflow port. Injecting the culture solution into the chamber and causing the culture solution to flow out of the outlet to form a cell tissue in the first region of the tissue formation region in the cell holding chamber. It is characterized by that.

In addition, the cells may be seeded in the range of 2 to 1.5 × 10 ⁵ cells per tissue body forming region.

  ADVANTAGE OF THE INVENTION According to this invention, while forming a cell tissue body in a short period, the microdevice which can culture the formed cell tissue body over a long period of time can be provided.

  Hereinafter, a cellular tissue microdevice according to an embodiment of the present invention will be described with reference to the drawings. The cell tissue microdevice according to the present invention is not limited to the following embodiment.

  FIG. 1 is an explanatory diagram of a cell tissue microdevice according to the present embodiment (hereinafter referred to as the present microdevice 1). As shown in FIG. 1, the microdevice 1 has a plurality of cell holding chambers 12 formed on a substrate 10 as depressions having a predetermined depth for holding cells.

  In the present microdevice 1, a culture solution in which cells are dispersed is put into the cell holding chamber 12, and the bottom surface 20 of the cell holding chamber 12 is used as the culture substrate surface of the cell, so that the cell tissue is transformed from the cell. Form the body.

  Here, as a cell for forming the cell tissue body, any cell species or organ / tissue type can be used as long as it forms a bond between cells. Specifically, as this cell, for example, primary cells collected from liver, pancreas, kidney, nerve, skin, etc. derived from animals such as humans, pigs, dogs, rats, mice, ES cells (Embryonic Stem cells), Established cell lines or cells obtained by genetic manipulation or the like can be used. In addition, as this cell, one type of cell can be used alone, or two or more types of cells can be mixed and mixed at an arbitrary ratio.

  Moreover, as a culture solution, an aqueous solution having any composition can be used as long as it is an aqueous solution containing necessary salts, nutrient components, and the like at appropriate concentrations so that the survival state and function of the cells to be used can be maintained. . Specifically, for example, as the culture solution, a basal medium such as DMEM (Dulbecco's Modified Eagle's Medium) added with antibiotics, so-called physiological saline, or the like can be used.

  Further, as will be described later, in the culture process in which a cell tissue body is formed from cells in the cell holding chamber 12, a culture solution is circulated in the cell holding chamber 12. For this reason, as shown in FIG. 1, the cell holding chamber 12 includes an inlet 22 for allowing the culture solution to flow into the cell holding chamber 12 and a flow for allowing the culture solution to flow out of the cell holding chamber 12. And an outlet 24.

  Further, as shown in FIG. 1, on the substrate 10 of the microdevice 1, an inflow path 14 communicating with each cell holding chamber 12 via an inlet 22 of each cell holding chamber 12, and each cell holding An outflow passage 16 communicating with each cell holding chamber 12 via the outflow port 24 of the chamber 12 is formed as a groove having a predetermined depth.

  As described above, in the microdevice 1 shown in FIG. 1, the cell holding chamber 12, the inflow path 14 for allowing the culture solution to flow into the cell holding chamber 12, and the culture solution flowing out from the cell holding chamber 12. A plurality of sets of outflow passages 16 are formed on the substrate 10 in parallel.

  Here, the substrate 10 of the present microdevice 1 is, for example, a synthetic resin such as polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, polyester (polyethylene terephthalate, etc.), polyurethane, polysulfone, polyacrylate, polymethacrylate, polyvinyl, silicon, or the like. It is made of a synthetic rubber such as EPDM (Ethylene Propylene Diene Monomer), or a metal material such as natural rubber, glass, ceramic, stainless steel, etc., and is formed as a plate-like body as shown in FIG.

  Further, the cell holding chamber 12, the inflow path 14, and the outflow path 16 can be formed by using any processing method selected according to the material of the substrate 10 and the like, for example, perforation using a machining center or the like, It can be formed on the substrate 10 by optical fine processing using laser or the like, etching processing, embossing or the like, or can be formed at the time of forming the substrate 10 by injection molding, press molding, stereolithography or the like.

  By such a processing method, the cell holding chamber 12, the inflow path 14, and the outflow path 16 are formed on the surface of the substrate 10 having a predetermined thickness, for example, a recess (bottomed hole) or a groove having a depth smaller than the substrate thickness. After forming through holes having shapes corresponding to the cell holding chamber 12, the inflow path 14, and the outflow path 16 on the substrate 10, another member is bonded to one side of the substrate 10. The bottom surface can be formed. In addition, as a member for forming this bottom surface, for example, a substrate, a film, or the like made of the same or different material as the substrate 10 in which the through hole is formed can be used.

  2 illustrates a part of the bottom surface 20 of one cell holding chamber 12 (region A indicated by a rectangular broken line in FIG. 1) among the plurality of cell holding chambers 12 included in the microdevice 1 shown in FIG. FIG.

  As shown in FIG. 2, the cell holding chamber bottom surface 20 surrounds one first region 32 exhibiting cell adhesion and the first region 32, and exhibits lower cell adhesion than the first region 32. A plurality of tissue body forming regions 30 including the second region 34 are included.

  The first region 32 of the tissue body formation region 30 has, for example, a cell-adhesive surface having a charged state and hydrophilicity / hydrophobicity suitable for cell adhesion in a culture solution. Here, the surface exhibiting cell adhesion is, for example, such that when the cell settles on the surface in the culture solution, the cell changes its shape from a spherical shape to a relatively flat shape. A surface that can be stretched and bonded.

  In addition, the second region 34 surrounding the first region 32 is, for example, a surface having a charged state or hydrophilicity / hydrophobicity that exhibits low cell adhesion compared to the surface of the first region 32 in the culture solution. Have That is, for example, when cells are adhered to the surface of the second region 34 and the surface of the first region 32, the cells adhered to the surface of the second region 34 Compared to the cells adhered to the surface, the extent of extension is small, and it shows a more spherical shape.

  Further, for example, when shear stress is applied to the cells adhered to the surface of the second region 34 and the cells adhered to the surface of the first region 32 by circulating a culture solution, the first The cells adhered to the surface of the second region 34 are less exposed to the surface of the second region 34 than the cells adhered to the first region 32 by a small shear stress load (ie, under a small culture medium flow rate). Detach from.

  The surface of the first region 32 and the surface of the second region 34 are obtained from a living body on the material surface of the substrate 10 exposed as the bottom surface 20 of the cell holding chamber 12 when the cell holding chamber 12 is formed, for example. Alternatively, it can be formed as a surface on which a synthesized cell adhesive substance or a derivative thereof is immobilized.

  Examples of the cell adhesive substance immobilized on the surface of the first region 32 and the surface of the second region 34 include cell surface molecules such as proteins existing in the cell membrane of cells to be used (for example, integrin and sugar chain receptor). ) Can be used.

  Specifically, for example, as a cell adhesive substance obtained from a living body, collagen, fibronectin, laminin and the like can be used, and as a derivative thereof, for example, an arbitrary functional group or a high group can be added to the cell adhesive substance. Those obtained by bonding molecular chains or the like (for example, covalently bonding using a condensation reaction or the like) can be used.

  In addition, for example, the synthesized cell adhesion substance includes a specific amino acid sequence exhibiting cell adhesion (for example, arginine / glycine / aspartic acid (so-called RGD) sequence) or a specific sugar chain sequence (for example, galactose side). A compound having a chain or the like can be used, and as a derivative thereof, a substance obtained by binding an arbitrary functional group or a polymer to the cell adhesive substance can be used.

  The cell adhesive substance or derivatives thereof obtained or synthesized from these living bodies can be obtained by, for example, drying an aqueous solution of the cell adhesive substance or the like on the cell holding chamber bottom surface 20, or the cell adhesive substance or the like. In an aqueous solution, a chemical reaction (for example, a condensation reaction between a carboxyl group and an amino group) is caused between the functional group of the cell adhesive substance and the functional group on the cell holding chamber bottom surface 20. It can be immobilized on the cell holding chamber bottom surface 20 by forming a covalent bond or the like.

  However, the surface of the second region 34 is formed so as to exhibit lower cell adhesion than the surface of the first region 32. That is, for example, when the same kind of cell adhesive substance is immobilized on the surface of the second region 34 and the surface of the first region 32, the surface of the second region 34 has the first region 32. The molecular density immobilized (the number of molecules of the cell adhesive substance immobilized per unit area of the surface) can be formed to be smaller than that of the surface.

  Specifically, in this case, for example, when the collagen is immobilized on the cell holding chamber bottom surface 20 by drying the collagen solution on the cell holding chamber bottom surface 20, collagen to be dried on the second region 34 is used. As the solution, a solution having a lower collagen concentration than the collagen solution dried on the first region 32 can be used.

  Further, for example, another type of cell adhesive substance having a lower cell adhesion than the cell adhesive substance immobilized on the surface of the first region 32 may be immobilized on the surface of the second region 34. it can.

  Specifically, in this case, for example, collagen that is known to have a relatively strong adhesion of primary hepatocytes to the first region 32 (high cell adhesion to the primary hepatocytes) is immobilized, and the second region Can immobilize laminin, which is known to adhere to the primary hepatocytes more weakly than collagen (low cell adhesion to primary hepatocytes).

  Further, the surface of the second region 34 can be formed, for example, as the material surface itself of the substrate 10 exposed as the bottom surface 20 of the cell holding chamber 12 when the cell holding chamber 12 is formed.

  Specifically, for example, when using a polystyrene substrate 10 and using cells that adhere strongly to the surface on which collagen is immobilized as compared with the polystyrene surface, collagen is applied to the surface of the first region 32. As the surface of the second region 34, the polystyrene surface itself can be used.

  Further, the first region 32 may be formed near the center of the tissue formation region 30. In this case, for example, as shown in FIG. 2, if a plurality of tissue body formation regions 30 are regularly arranged at predetermined intervals on the cell holding chamber bottom surface 20, they are formed on the tissue body formation region 30. The cell tissue body can also be regularly arranged on the bottom surface 20 of the cell holding chamber, and the shape of the formed cell tissue body can be made uniform.

  Therefore, for example, in the case where each cell tissue is subjected to a staining treatment using a fluorescent dye or the like, and the function of each cell tissue is evaluated by the degree of staining, etc., each of the uniform cell tissue Since the position can be accurately specified by the coordinate value set on the microdevice 1 or the like, the degree of staining can be easily, quickly and reliably analyzed using an automatic analyzer or the like.

In addition, the diameter of the tissue body formation region 30 is preferably in the range of about 2 to 50 times the diameter of the cells to be used, and particularly preferably in the range of about 4 to 30 times. That is, the area of the tissue body formation region 30 cannot be generally described because the cell size varies depending on the type and state thereof, but is preferably in the range of, for example, 100 to 1 × 10 ⁶ μm ² , In particular, the range of 300 to 3 × 10 ⁵ μm ² cm ² is preferable.

  This is because the cells seeded on the tissue body formation region 30 gather to form a cell tissue body on the tissue body formation region 30, and therefore the size of the tissue body formation region 30 is 30 defines the number of cells contained in the cell tissue formed on the cell 30. When the size is smaller than the lower limit, the cell tissue is formed on the tissue body formation region 30. When the necessary number of cells cannot be adhered and the size is larger than the upper limit, the number of cells adhered on the tissue formation region 30 is too large, and thus a huge cellular tissue is formed. This is because the cells that are formed and located inside the cell tissue body cannot receive sufficient nutrients and oxygen from the culture solution outside the cell tissue body and may die.

  In addition, the shapes of the tissue body formation region 30, the first region 32, and the second region 34 are not particularly limited, and may be, for example, a circular shape as shown in FIG. It may be a polygon or the like.

  In addition, as shown in FIG. 2, the cell holding chamber bottom surface 20 includes a third region 36 surrounding each tissue body forming region 30. The third region 36 may be formed so as to exhibit lower cell adhesion than the second region 34 of each tissue body forming region 30. In this case, the third region 36 has, for example, a cell adhesion surface having a charged state or hydrophilicity / hydrophobicity that exhibits lower cell adhesion than the second region 34 in the culture solution.

  That is, for example, the surface of the third region 36 can be formed as the material surface itself of the substrate 10 exposed as the bottom surface 20 of the cell holding chamber 12 when the cell holding chamber 12 is formed, or is exposed. It can be formed on the surface of the substrate 10 as a surface on which a cell adhesive substance obtained or synthesized from a living body or a derivative thereof is immobilized.

  The surface of the third region 36 is, for example, similar to the case where the surface of the second region 34 described above is formed as a surface exhibiting cell adhesion lower than the surface of the first region 32. When the same kind of cell adhesive substance is immobilized on the surface and the surface of the second region 34, the molecule immobilized on the surface of the third region 36 as compared with the surface of the second region 34. It can be formed so as to have a low density, or the surface of the third region 36 has another kind of cell adhesiveness lower than that of the cell adhesive substance immobilized on the surface of the second region 34. It can be formed by immobilizing the cell adhesive substance.

  The third region 36 may be formed as having a non-cell-adhesive surface. In this case, the surface of the third region 36 is, for example, a cell non-adhesive having a charged state or hydrophilicity / hydrophobicity inappropriate for cell adhesion such that cells cannot substantially adhere in the culture solution. Can be formed as a surface. Here, the surface showing cell non-adhesiveness means that, for example, when cells settle on the surface in a culture solution, the cell shape adheres very weakly to such an extent that the cell shape hardly changes from a spherical shape. It is a surface that can be easily detached by the flow of the liquid or the like, or the cells cannot adhere at all and remain floating in the culture medium in a spherical shape.

  In this case, the surface of the third region 36 is obtained from a living body or synthesized on the material surface of the substrate 10 exposed as the bottom surface 20 of the cell holding chamber 12 when the cell holding chamber 12 is formed, for example. It can be formed as a surface on which a cell non-adhesive substance or a derivative thereof is immobilized.

  As the cell non-adhesive substance immobilized on the third region 36, for example, a cell non-adhesive substance derived from a living body or synthesized that does not bind to cell surface molecules such as proteins and sugar chains existing in the cell membrane of the cell to be used. Substances or their derivatives can be used.

  Specifically, for example, as a cell-non-adhesive substance derived from a living body, a highly hydrophilic protein such as albumin can be used, and as a derivative thereof, for example, any cell non-adhesive substance can be used. Those having a functional group or a polymer chain bonded thereto can be used.

  In addition, for example, as the synthesized cell non-adhesive substance, polymers having extremely high hydrophilicity such as polyethylene glycol, MPC (2-methacryloyloxyethyl phosphorylcholine), poly-HEMA (polyhydroxyethyl methacrylate), SPC ( Segmented polyurethane) and the like can be used, and as these derivatives, those obtained by binding any functional group or polymer to the cell non-adhesive substance can be used.

  The cell non-adhesive substance or derivative thereof obtained or synthesized from these living bodies can be obtained by, for example, drying an aqueous solution of the cell non-adhesive substance or the like on the cell holding chamber bottom surface 20 or the cell non-adhesive substance. In an aqueous solution of a substance or the like, the cell retention is achieved by causing a chemical reaction between the functional group of the cell non-adhesive substance or the like and the functional group on the cell holding chamber bottom surface 20 to form a covalent bond. It can be immobilized on the bottom surface 20 of the chamber.

  The cell non-adhesive third region 36 is formed so as to surround each tissue body forming region 30, so that cells are selected on each tissue body forming region 30 in the cell holding chamber bottom surface 20. Adhesion can be made easy. In addition, the third region 36 is not limited to be formed so as to surround each tissue body forming region 30. For example, the third region 36 includes a plurality of tissue body forming regions 30 on the cell holding chamber bottom surface 20. It may be formed so as to be separated from each other.

  Moreover, the cell holding chamber bottom surface 20 of the microdevice 1 may include a cell holding cavity 40 formed as a bottomed hole. In this case, for example, as shown in FIG. 3, the cell holding cavity 40 has a bottom surface 42 and a side surface 44 that form the pore structure, and the cell holding cavity bottom surface 42 has one tissue body forming region 30. Can be formed.

  The cell holding cavity 40 can be formed by using an arbitrary processing method selected according to the material of the substrate 10, for example, drilling using a machining center or the like, optical fine processing using a laser or the like, It can be formed on the cell holding chamber bottom surface 20 by etching, embossing or the like, or can be formed at the time of molding the cell holding chamber 12 by injection molding, press molding, stereolithography or the like.

  Further, the cell holding cavity bottom surface 42 is configured so that the whole cell holding cavity bottom surface 42 becomes the tissue body forming region 30 (that is, not including the third region 36 surrounding the tissue body forming region 30 as shown in FIG. 3). ) Can also be formed. In this case, for example, by forming the cell holding cavity 40 on the cell holding chamber bottom surface 20 by drilling using a machining center or the like, the material surface itself of the substrate 10 exposed as the bottom surface 42 of the cell holding cavity 40 can be used as the second. A first region 32 in which a cell adhesive substance is immobilized can be formed near the center of the second region 34 as the surface of the region 34. In this way, the tissue formation region 30 having a predetermined size can be easily formed on the cell holding chamber bottom surface 20.

  In addition, the cell holding cavity bottom surface 42 is preferably formed in an appropriate size range as the tissue body forming region 30. That is, the diameter of the cell holding cavity bottom surface 42 is preferably in the range of about 2 to 50 times the diameter of the cells to be used, and particularly preferably in the range of about 4 to 30 times. Further, the shape of the cell holding cavity bottom surface 42 is not particularly limited, and may be, for example, a circular shape as shown in FIG. 3, or may be an elliptical shape, a polygonal shape, or the like.

  Further, the depth of the cell holding cavity 40 is preferably in the range of about 1 to 50 times the diameter of the cell to be used, and particularly preferably in the range of about 2 to 30 times. This is because when the depth of the cell holding cavity 40 is smaller than the lower limit value, it becomes difficult to securely hold the cells in the cell holding cavity 40, and when the depth is larger than the upper limit value. This is because supply of oxygen and nutrients to the cells on the cell holding cavity bottom surface 42 may be insufficient.

  Further, the cell holding cavity 40 can be formed so as to be regularly arranged at a predetermined interval on the cell holding chamber bottom surface 20 in the same manner as the plurality of tissue body forming regions 30 shown in FIG. In this case, the plurality of regularly arranged cell holding cavities 40 can be formed by using, for example, a machining center or the like that precisely controls a processing position by a CAD (Computer Aided Design) program.

  In addition, as shown in FIG. 4, in the microdevice 1, a plurality of cell holding chambers 12 are connected in series via a channel on a substrate 10, and a set of the plurality of cell holding chambers 12 and the channel is combined. However, a plurality of them may be formed in parallel.

  In this case, for example, by forming each cell holding chamber bottom surface 20 as including one tissue body forming region 30, one cell tissue body can be formed and held on each cell holding chamber bottom surface 20. .

  Further, the cell holding chamber 12 shown in FIG. 4 is, for example, a cavity shape that is further depressed from a flow path that communicates with the cell holding chamber 12 formed on the substrate 10 like the cell holding cavity 40 shown in FIG. May be formed.

  Next, an outline of a method for forming and culturing a cell tissue body using the present microdevice 1 will be described. First, the cells to be used are dispersed in the culture solution so as to have a predetermined density, and a predetermined amount of the cell dispersion solution is placed in each cell holding chamber 12 to seed the cells.

  Here, the number of cells to be seeded in one cell holding chamber 12 can contact the cells settled on the cell holding chamber bottom surface 20 to the extent necessary to form a bond with each other, and the cells It is preferable to determine so that the size of the cell tissue formed by gathering can be suppressed within a predetermined range.

Specifically, for example, the number of seeded cells is preferably in the range of 2 to 1.5 × 10 ⁵ per tissue body forming region 30 on the cell holding chamber bottom surface 20, particularly 50 to 3.0. The range of × 10 ⁴ is preferable. The number of seeded cells per unit area of each tissue body formation region 30 is preferably in the range of 30 to 1.5 × 10 ⁴ cells / mm ² .

  This is because, in order to form a cell tissue body, it is necessary to hold at least two cells per tissue body formation region 30, and if the number of seeded cells is too large, cells formed from the cells This is because the tissue becomes huge and the cells inside it may be necrotic due to lack of nutrients and oxygen.

  Next, the microdevice 1 in which cells are seeded in this manner is kept horizontal and kept stationary for a predetermined period of time, so that the cells are settled on the cell holding chamber bottom surface 20 and cultured. . In the stationary culture process, among the cells that have settled on the cell holding chamber bottom surface 20, the cells that have settled on the first region 32 of the tissue body forming region 30 adhere relatively strongly to the surface of the first region 32. The cells that have settled on the second region 34 adhere to the surface of the second region 34 with a weaker adhesive force than the cells that have settled on the first region 32. In addition, the cells that have settled on the third region 36 adhere very weakly to the surface of the third region 36 or float in the culture solution without adhering.

  Then, after the cells are adhered to at least the surface of the first region 32 and the surface of the second region 34 by this stationary culture, the culture solution is introduced at a predetermined flow rate from the inlet 22 of each cell holding chamber 12. Then, the flow of the culture solution in each cell holding chamber 12 is started by causing the culture solution to flow out from the outlet 24 of each cell holding chamber 12 at the predetermined flow rate.

  Here, the flow rate of the culture solution is such that, for example, the cells adhered to the first region 32 and the cells adhered to the second region 34 are not detached from the adhesion surface, and the cells on the third region 36 are removed. It can be experimentally determined as a flow rate in a range to be detached, or as a flow rate in a range in which cells adhering to the first region 32 are not desorbed and the cell adhering to the second region 34 is promoted.

  In this flow-through culture process, as the culture time elapses, cells adhered on the first region 32, cells adhered on the second region 34, cells adhered on the first region 32 and the first The cells adhered on the two regions 34 form an intercellular bond, and the cells adhered on the second region 34 gradually desorb from the surface of the second region 34 while maintaining the intercellular junction. Then, the cells gather on the first region 32, and after a predetermined culture period, a cell tissue body in which the cells are three-dimensionally connected is formed on the first region 32.

  Thus, by using this microdevice 1, a cell tissue body can be formed within a short period of time after the start of culture. This is because the culture state of the cells can be kept good by continuously supplying a culture solution containing sufficient nutrients and oxygen to the cells in the cell holding chamber 12, and the culture solution. This is considered to be due to the fact that the shear stress is applied near the cell surface by the flow of the cell to promote the desorption of cells that are relatively weakly adhered to the second region 34.

  In addition, the cell tissue formed in this way on the first region 32 of each tissue body formation region 30 does not float in the culture medium through the stationary culture period and the flow culture period, and the first The culture can be performed over a long period of time while being stably held in a state of being adhered on the region 32.

  Also, as shown in FIGS. 1 and 4, by forming a plurality of cell holding chambers 12 and flow paths in parallel on the substrate 10 of the microdevice 1, for example, different drugs can be applied to each set. Since the culture medium containing it can be distributed, it becomes possible to perform various analyzes and evaluations using the cell tissue bodies formed in each group.

[Example]
Next, an example in which a cell tissue body is formed using the present microdevice 1 will be described.

[Production of cell tissue microdevice]
In this example, a flat plate made of polymethyl methacrylate (24 mm × 24 mm, thickness 400 μm) was used as the substrate 10 of the microdevice 1. That is, the bottom surface 20 and the inlet 22 as shown in FIG. 5 are formed on a part of the surface of the polymethyl methacrylate flat plate by a milling process using a machining center (desk type NC fine processing machine, manufactured by PMT Co., Ltd.). A cell holding chamber 12 having an outlet 24, a liquid inlet 15 that is open at one end, an inlet 14 that communicates with the inlet 22 at the other end, and a liquid outlet that is open at one end. 17 and one outflow passage 16 having the other end communicating with the outflow port 24 was formed. As shown in FIG. 5, the cell holding chamber 12 includes a rectangular region having a longitudinal length L1 of 5 mm and a width W1 of 5 mm, and a Taber region having a length L2 of 2.5 mm connected to both longitudinal ends of the rectangular region. And a depth of 200 μm. By providing this tapered region, the flow rate distribution in the width direction of the cell holding chamber of the culture solution flowing into the cell holding chamber 12 from the inlet 22 can be easily made relatively uniform. it can. Further, as shown in FIG. 5, the inflow path 14 and the outflow path 16 communicating with the cell holding chamber 12 were formed to have a width W2 of 300 μm and a depth of 200 μm.

  Further, a diameter for forming a side surface 44 (height: 200 μm) of the cell holding cavity 40 as shown in FIG. 3 by subjecting a part of the cell holding chamber bottom surface 20 to a drilling process using a machining center over a rectangular area of 5 mm. About 250 300 μm circular through holes were formed. The circular through-holes were regularly arranged so that the distance between the centers was 400 μm. A thin film (thickness 9 nm) of platinum (Pt) is formed on the bottom surface 20 of the cell holding chamber in which the through holes are formed by performing a sputtering process using a sputtering apparatus (E-1030, manufactured by Hitachi, Ltd.). Formed.

Next, as a cell non-adhesive substance immobilized on the surface of the third region 36, a cell non-adhesive synthetic polymer having a polyethylene glycol (PEG) chain with a molecular weight of 5000 (chemical formula: CH ₃ (CH ₂ CH ₂ )) _n SH, manufactured by Nektar Therapeutics). Then, the cell holding chamber bottom surface 20 on which the platinum thin film is formed is immersed in an ethanol solution containing the cell non-adhesive polymer at a concentration of 25 mg / mL, and the thiol possessed by the cell non-adhesive polymer in a nitrogen atmosphere. The cell non-adhesive polymer was immobilized on the platinum surface by forming a chemical bond between the group and the platinum surface of the cell holding chamber bottom surface 20. Thereafter, the entire cell holding chamber bottom surface 20 was sufficiently washed with an ethanol solution to remove excess cell non-adhesive polymer.

  Further, a glass flat plate (22 mm × 22 mm, thickness 400 μm) was used as the bottom surface 42 of the cell holding cavity 40 of the microdevice 1. That is, this glass flat plate and the portion where the through-hole is formed on the polymethyl methacrylate flat plate are aligned, and the glass flat plate and the poly-polyethylene are obtained using a silicon-based adhesive (TSE389, manufactured by GE Toshiba Silicone Co., Ltd.). A methyl methacrylate flat plate was adhered. In this manner, a cell holding cavity 40 having a depth of 200 μm and a circular bottom surface 42 having a diameter of 300 μm as shown in FIG. 3 was formed.

  On the other hand, a stamp made of PDMS (Poly (Dimethyl Siloxane)) having a plurality of cylindrical protrusions having a diameter of 100 μm and a length of 300 μm was produced by molding. In this stamp, the center-to-center distance of the circular cross section of the tip of the protrusion was set to 400 μm so that the position of each cylindrical protrusion corresponded to the position of each cell holding cavity 40 on the cell holding chamber bottom surface 20. Then, by microcontact printing using this stamp, the first region 32 as shown in FIG. 3 was formed on the cell holding cavity bottom surface 42 as follows.

  That is, collagen (Cellmatrix Type IC, manufactured by Nitta Gelatin Co., Ltd.) was prepared as a cell adhesive substance to be immobilized on the surface of the first region 32. And the collagen solution was apply | coated to the front end surface of each said cylindrical protrusion by immersing the front-end | tip of each cylindrical protrusion of the said produced stamp in the aqueous solution which contains this collagen with the density | concentration of 0.3 mg / mL.

  Next, while observing under a phase contrast microscope, the position of each cylindrical projection of the stamp coated with the collagen solution is aligned with the vicinity of the center of each cell holding cavity bottom surface 42, and the tip of each cylindrical projection is aligned The collagen solution applied to the tip of each cylindrical protrusion was applied to the vicinity of the center of each cell holding cavity bottom surface 42 by pressing on each cell holding cavity bottom surface 42. Thereafter, the collagen solution applied to the cell holding cavity bottom surface 42 was dried, and the collagen was fixed onto the cell holding cavity bottom surface 42.

  In this way, one first region 32 having a diameter of about 100 μm and having collagen immobilized thereon was formed near the center of the bottom surface 42 of each cell holding cavity having a diameter of 300 μm. Further, a portion other than the first region 32 in each cell holding cavity bottom surface 42, that is, the glass flat plate surface itself was used as the second region 34. That is, on each cell holding cavity bottom surface 42, there is a tissue body forming region 30 that includes a first region 32 and a second region 34, and the first region 32 is formed near the center of the second region 34. One was formed.

  Furthermore, glass is formed on the upper surface of the substrate 10 on which the cell holding chamber 12, the inflow path 14, and the outflow path 16 are formed (the surface opposite to the side on which the glass plate forming the cell holding cavity bottom surface 42 is bonded). A manufactured flat plate (24 mm × 24 mm, thickness 400 μm) was adhered using a silicon-based adhesive to form an upper lid covering the cell holding chamber 12, the inflow path 14, and the outflow path 16.

[Production of culture system]
Next, a culture system for perfusing a culture solution into the cell holding chamber 12 of the microdevice 1 thus manufactured was prepared. That is, a silicon tube (inner diameter 0.5 mm, outer diameter 1.0 mm) having one end inserted into the glass bottle so that the culture solution (30 mL) held in the glass bottle can be perfused into the cell holding chamber 12. And the other end of another silicon tube having one end inserted into the glass bottle and the other end of the other silicon tube connected to the cell holding chamber 12. A part of the silicon tube was set in a pump (micro tube pump, manufactured by EYELA Co., Ltd.) connected to the liquid supply / outflow port 17 of the outflow passage 16 communicating with the holding chamber 12. That is, in the perfusion culture system including the microdevice 1, the culture medium in the glass bottle is connected to the liquid supply inlet 15 of the inflow path 14 communicating with the cell holding chamber 12 by driving the pump. Into the cell holding chamber 12 at a predetermined flow rate, and from the cell holding chamber 12 through the silicon tube connected to the liquid supply / outflow port 17 of the outflow passage 16 communicating with the cell holding chamber 12. Was flown out at the predetermined flow rate, and returned to the glass bottle again, so that the culture solution could be cultured while being perfused.

  Further, as a micro device for a control experiment, a cell holding chamber 12, an inflow path 14 and an outflow path 16 as shown in FIG. 5 are formed on a polymethyl methacrylate flat plate (24 mm × 24 mm, thickness 400 μm), In addition, without forming the cell holding cavity 40 on the bottom surface 20 of the cell holding chamber 12, the whole cell holding chamber bottom surface 20 is coated with collagen (Cellmatrix Type I-C, manufactured by Nitta Gelatin Co., Ltd.). Then, similarly to the present microdevice 1, a culture system for perfusing a culture solution into the cell holding chamber 12 of the control experimental microdevice was produced.

  The perfusion culture system thus prepared was sterilized by an alcohol solution and ultraviolet irradiation for 6 hours and used for the following culture experiments.

[Preparation of cells]
In this example, primary hepatocytes prepared from rat liver using a known collagenase perfusion method were used as cells. Here, the preparation method of this hepatocyte is demonstrated easily. That is, first, a cannula is inserted into the portal vein (one of blood vessels connected to the liver) of a 7-week-old Wistar rat (body weight 250 g), blood removal of a predetermined composition is introduced into the liver from the cannula, and collagenase is then introduced. The digestion solution which melt | dissolved (made by Wako Pure Chemical Industries) at the density | concentration of 0.5 mg / mL was perfused, and the digestion process which cut | disconnects the coupling | bonding etc. of hepatocytes in a liver was performed. Then, the digested liver was extracted, and dispersed hepatocytes were obtained by a dispersion treatment using a scalpel or a pipette and a washing treatment using a culture solution or the like.

  As the culture solution, Dulbecco's modified Eagle medium (DMEM, Gibco) 13.5 g / L, 60 mg / L proline (Sigma), 50 mg / mL epidermal growth factor (EGF, manufactured by Funakoshi) ), 7.5 mg / L hydrocortisone (manufactured by Wako Pure Chemical Industries, Ltd.), 0.1 μM copper sulfate pentahydrate (manufactured by Wako Pure Chemical Industries, Ltd.), 3 μg / L selenic acid (manufactured by Wako Pure Chemical Industries, Ltd.), 50 pM zinc sulfate heptahydrate (Wako Pure Chemical Industries), 50 μg / L linoleic acid (Sigma), 58.8 mg / L penicillin (Meiji Seika), 100 mg / L streptomycin (Meiji) Confectionery), 1.05 g / L sodium bicarbonate (Wako Pure Chemical Industries), 1.19 g / L 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES, Made by Dojindo) Was used serum-free culture medium were added.

[Formation of cell tissue]
The hepatocytes thus obtained were dispersed in the culture solution so as to have a density of 5.0 × 10 ⁵ cells / mL. Then, 100 μL of a part of this cell dispersion is added into the cell holding chamber 12 of the microdevice 1 after the sterilization treatment, and left still at 37 ° C. in an atmosphere of 5% carbon dioxide gas and 95% air. Culture was performed for 2 hours. Further, 100 μL of another part of this cell dispersion was added into the cell holding chamber 12 of the microdevice for control experiment, and static culture was performed in the same manner as in the microdevice 1.

  Next, after confirming that the hepatocytes settled on the bottom surface 42 of each cell were adhered to the first region 32 and the second region 34 under a phase contrast microscope, the pump was driven, and the cell retention chamber was Perfusion of the culture medium in 12 was started. The flow rate of the culture solution in this perfusion culture was 1 mL / h.

  Also, in the control experimental microdevice, at the start of circulation of the culture solution in the microdevice 1, the adhesion of hepatocytes on the cell holding chamber bottom surface 20 (collagen coating surface) is confirmed, and the culture in the culture vessel is performed. Fluid perfusion was started.

  FIG. 6 is a phase contrast micrograph of the hepatocyte tissue body (T in FIG. 6) formed in the cell holding cavity 40 according to a part of the cell holding chamber bottom surface 20 of the microdevice 1 on the third day of culture. Show. As shown in FIG. 6, one hepatocyte tissue body was formed in each cell holding cavity 40, and the shape thereof was a spherical shape whose surface was smoothed. Such a spherical hepatocyte tissue body was formed within 1 to 2 days after the start of culture in each cell holding cavity 40 of the present microdevice 1.

  Further, as shown in FIG. 6, each hepatocyte tissue body was formed in the vicinity of the center of each cell holding cavity bottom surface 42 in each cell holding cavity 40. That is, each hepatocyte tissue body is formed on the first region 32 in each cell holding cavity bottom surface 42 (that is, the tissue body forming region 30), and maintains an adhesive state on the first region 32. It was stably maintained without floating in the culture medium throughout the perfusion culture period over a week. Note that almost no hepatocytes were present on the second region 34 of the bottom surface 42 of each cell holding cavity. On the other hand, in the control experiment, hepatocytes were adhered in a monolayer state (not shown) extending flat on the cell holding chamber bottom surface 20 coated with collagen.

  Moreover, albumin (one of blood proteins in the living body), which is one of the detoxifying functions peculiar to the liver, with respect to the hepatocyte tissue formed in the present microdevice 1 and the hepatocytes in the monolayer state in the control experiment. ) Secretory capacity was evaluated. In the evaluation of the albumin secretion ability of hepatocytes, on the third, seventh, tenth and fourteenth days after the start of culture, the increase in the concentration of albumin contained in the perfused culture solution within 24 hours is determined by a known ELISA. The measurement was performed using the (Enzyme Linked ImmunoSorbent Assay) method.

FIG. 7 shows the evaluation results of the albumin secretion ability. In FIG. 7, the horizontal axis represents the culture time (culture days), and the vertical axis represents the amount of albumin secretion (μg) per unit time (hour) per unit cell number (10 ⁶ cells). In FIG. 7, circles indicate the results for the hepatocyte tissue formed using the present microdevice 1, and squares indicate the results for the monolayered hepatocytes in the control experiment.

  As shown in FIG. 7, the hepatocyte tissue formed in the present microdevice 1 continued to express albumin secreting ability at a higher level over two weeks of culture than the monolayered hepatocytes in the control experiment. In contrast, as shown in FIG. 7, the albumin secretion ability of the monolayered hepatocytes in the control experiment monotonously decreased with the progress of culture after the start of culture, and almost disappeared at the time of 2 weeks of culture.

  As described above, by using the present microdevice 1, a hepatocyte tissue that continuously expresses a liver-specific function at a high level is formed from a hepatocyte in a short period of time, and a hepatocyte tissue that expresses the high liver function. The body could be cultured for a long period of time while being held on the bottom surface 42 of each cell holding cavity 40.

It is explanatory drawing about the cell tissue microdevice which concerns on one Embodiment of this invention. It is explanatory drawing about the structure | tissue formation area | region which concerns on one Embodiment of this invention. It is explanatory drawing about the cell holding | maintenance cavity which concerns on one Embodiment of this invention. It is explanatory drawing about the cell tissue microdevice which concerns on other embodiment of this invention. It is explanatory drawing about the cell holding chamber which concerns on one Embodiment of this invention. 3 is a phase contrast micrograph of a hepatocyte tissue formed using the cell tissue microdevice according to an embodiment of the present invention. It is a graph which shows the evaluation result of the albumin secretion ability of the hepatocyte structure | tissue formed using the cell structure | tissue microdevice which concerns on one Embodiment of this invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 Cell organization microdevice, 10 board | substrate, 12 cell holding | maintenance chamber, 14 inflow path, 15 liquid supply inflow port, 16 outflow path, 17 liquid supply outflow port, 20 cell holding chamber bottom face, 22 inflow port, 24 outflow port, 30 Tissue formation region, 32 1st region, 34 2nd region, 36 3rd region, 40 cell retention cavity, 42 cell retention cavity bottom, 44 cell retention cavity side.

Claims (9)

Having a cell holding chamber for holding cells;
The cell holding chamber includes a bottom surface including at least one tissue body forming region, an inlet for allowing a culture solution to flow into the cell holding chamber, and an outlet for allowing the culture solution to flow out from the cell holding chamber. Have
The tissue forming area surrounds one of the first region showing cell adhesiveness, the first region, seen including a second region indicating the low cell adhesion as compared to the first region, the area that Is in the range of 100 to ¹ × 10 ⁶ μm ² . The bottom surface of the cell holding chamber includes a third region that surrounds the tissue formation region and exhibits lower cell adhesion than the second region.
The cellular tissue body microdevice according to claim 1. The bottom surface of the cell holding chamber includes a plurality of the tissue body forming regions, and includes a third region that separates the plurality of tissue body forming regions from each other and exhibits low cell adhesion compared to the second region.
The cellular tissue body microdevice according to claim 1. The cell holding chamber bottom includes a cell holding cavity,
The bottom surface of the cell holding cavity includes one tissue body formation region,
The cellular tissue microdevice according to any one of claims 1 to 3, wherein The first region is formed near the center of the tissue formation region,
The cellular tissue microdevice according to any one of claims 1 to 4, wherein In the first region, a cell adhesive substance obtained from a living body or synthesized, or a derivative thereof is immobilized,
The cellular tissue body microdevice according to any one of claims 1 to 5, wherein A cell tissue is held in the first region,
The cellular tissue microdevice according to any one of claims 1 to 6, Seeding cells forming a bond with each other in the cell holding chamber of the cell tissue microdevice according to any one of claims 1 to 7,
From the inflow port, a culture solution flows into the cell holding chamber seeded with the cells, and the culture solution flows out from the outflow port, so that the tissue formation region in the cell holding chamber is in the first region. Forming a cellular tissue in a region;
A method of forming a cell tissue, comprising: The cells are seeded in the range of 2 to 1.5 × 10 ⁵ cells per each tissue formation region,
The method for forming a cell tissue body according to claim 8.

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