WO2018070441A1 - Method for producing culture cell structure - Google Patents

Abstract

Provided is a production method for efficiently obtaining a three-dimensional structure of a culture cell by culturing a laminated cell sheet. This method for producing a culture cell structure comprises: placing a cell sheet laminate, in which two or more cell sheets are laminated, on the surface of a cell placement part of a substrate disposed in a container for accommodating a liquid culture medium; and culturing, in the liquid culture medium, the cell sheet laminate that is on the cell placement part of the substrate, wherein the substrate has a recessed portion in the rear surface of the cell placement part, and is provided with at least one fiber passing through the cell placement part in the direction of the recessed portion, and the cell sheet laminate is cultured while the corresponding fiber is extended in the lamination direction.

Description

Method for producing cultured cell structure

The present invention relates to a method for producing a cultured cell structure.

In recent years, cell transplantation treatment using a cell sheet has been studied as a medical treatment for replenishing cells to a tissue deficient due to disease, damage or the like. For example, production of tissues such as kidneys and livers using a plurality of cell sheets has been studied. Since these tissues have a complicated three-dimensional structure and various types of cells are organized, it is difficult to organize them only by culturing the cells. Therefore, a method for producing a three-dimensional tissue in which formation of an extracellular matrix and culturing of cells thereon are repeated has been proposed (see, for example, Patent Document 1). In addition, a method for producing a laminated cell sheet by superposing cell sheets obtained by two-dimensionally growing cells has been proposed (see, for example, Patent Document 2).

JP 2007-228921 A International Publication No. 2014/192909

An object of the present invention is to provide a production method for efficiently obtaining a three-dimensional structure of cultured cells by culturing stacked cell sheets.

Specific means for solving the above problems are as follows.
Placing a cell sheet laminate in which two or more cell sheets are laminated on a cell placement part surface of a substrate placed in a container containing a liquid medium; and on the cell placement part of the substrate The cell sheet laminate is cultured in a liquid medium, and the substrate has a recess on the back surface of the cell mounting portion, and the substrate has the cell mounting portion facing the recess. A method for producing a cultured cell structure, comprising at least one fiber penetrating, and culturing the cell sheet laminate in a state in which the fiber extends in the lamination direction.

According to the present invention, it is possible to provide a production method for efficiently obtaining a three-dimensional structure of cultured cells by culturing stacked cell sheets.

It is a schematic sectional drawing which shows typically an example of the cultivation method of the cell sheet laminated body which concerns on this embodiment. It is a figure which shows the expression level of the cell proliferation marker in a cultured cell structure. It is a figure which shows the expression level of the cell respiration marker in a cultured cell structure. It is a figure which shows the expression level of the cell respiration marker in a cultured cell structure. It is a figure which shows an example of the image which immunostained the hypoxia marker in the cultured cell structure which concerns on Example 1. FIG. It is a figure which shows an example of the image which immunostained the hypoxia marker in the cultured cell structure which concerns on the comparative example 1. FIG. It is a figure which shows an example of the image which immunostained the cell proliferation marker in the cultured cell structure which concerns on Example 1. FIG. It is a figure which shows an example of the image which immunostained the cell proliferation marker in the cultured cell structure which concerns on the comparative example 1. FIG. It is a figure which shows an example of the image which immunostained the cell respiration marker in the cultured cell structure which concerns on Example 1. FIG. It is a figure which shows an example of the image which immunostained the cell respiration marker in the cultured cell structure which concerns on the comparative example 1. FIG. It is a figure which shows the expression level of the cell respiration marker in the cultured cell structure in connection with the comparative example 2, Example 5, and Example 6. FIG. It is a figure which shows an example of the SEM image of the hollow filament obtained by coating CYTOP CTL-109S on the syringe needle with an internal diameter of 400 micrometers. It is a figure which shows an example of the SEM image of the hollow filament obtained by pre-coating gelatin film as a sacrificial layer on the outer surface of a syringe needle having an inner diameter of 400 μm, coating Flemion EW909, and annealing in an oven at 120 ° C. for 10 minutes. .

In the method for producing a cultured cell structure according to the present invention, a cell sheet laminate in which two or more cell sheets are laminated is placed on the surface of a cell placement portion of a substrate placed in a container that contains a liquid medium. And culturing the cell sheet laminate on the cell placement part of the base material in a liquid medium, the base material has a recess on the back surface of the cell placement part, and the base material is The cell sheet laminate is cultivated with at least one fiber penetrating the cell placement portion toward the recess, and the fiber extends in the stacking direction. By culturing the cell sheet laminate on a substrate placed in a liquid medium with fibers, the cell sheet laminate can be efficiently cultured, and a three-dimensional cultured cell comprising cultured cells A structure can be manufactured efficiently. In particular, it is preferable that at least one of the fibers penetrates the cell sheet laminate. This can be considered to be because, for example, oxygen and nutrients are efficiently supplied into the cell sheet laminate through fibers.

In the method for producing a cultured cell structure, a cell sheet laminate in which two or more cell sheets are laminated is placed on the surface of a cell placement portion of a substrate placed in a container that contains a liquid medium. The size, shape, and material of the container for storing the liquid medium are not particularly limited as long as the liquid medium and the substrate can be disposed. Examples of the container include a polystyrene petri dish having a diameter of 3.5 to 15 cm, a 6-well plate, and the like.

Liquid Medium The liquid medium is prepared using a medium selected according to the cell type constituting the cell sheet laminate to be cultured as a basal medium. As the basal medium, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dolbecco's modified Eagle's Medium (DMEM) medium, Ham's F16h medium, RP 's medium and mixed medium thereof can be mentioned.

Serum may be contained in the liquid medium. The liquid medium may be, for example, albumin, transferrin, sodium selenite, ITS-X (Invitrogen) (containing insulin, transferrin, sodium selenite), Knockout Serum Replacement (KSR) (when ES cells are cultured). FBS serum replacement), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen precursor, trace element, 2-mercaptoethanol, 3'-thiolglycerol, etc. May be included. Furthermore, one or more selected from lipids, essential amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, low molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, etc. An additive may be included.

The base material on which the cell sheet laminate is placed has at least a cell placement portion, and has a recess on the back surface of the cell placement portion. Examples of the shape of the cell placement portion include a rectangular shape, a polygonal shape such as a triangular shape, a substantially circular shape, and an elliptical shape. What is necessary is just to select the magnitude | size of a cell mounting part suitably according to the magnitude | size of the cell sheet laminated body to culture | cultivate, the magnitude | size of a container, etc. FIG. For example, in the case of a substantially circular shape, the size of the cell placement portion is 0.5 cm or more in diameter, and preferably 1 cm or more. Moreover, it is 10 cm or less, Preferably it is 5 cm or less.

The thickness of the base material, that is, the height in the direction perpendicular to the surface of the cell placement portion is appropriately selected according to the height of the container and the like, for example, 0.1 mm or more and 20 mm or less, preferably 1 mm or more and 15 mm or less. is there.

The back surface of the cell placement portion of the base material may have a wall portion that forms a recess together with the back surface of the cell placement portion. The end portion of the wall portion opposite to the cell placement portion may be in contact with the bottom surface of the inner wall of the container or may be spaced apart. It is preferable that at least a part of the end portion of the wall portion of the base material is in contact with the bottom surface of the inner wall of the container.

The size of the concave portion is, for example, 0.1 cm to 10 cm, and preferably 1 cm to 5 cm, as the height of the wall portion forming the concave portion. The wall thickness is, for example, 0.1 mm to 5 mm, preferably 1 mm to 2 mm.

Examples of the base material include polystyrene (PS), polyethylene (PE), polyethylene terephthalate (PET), nylon, polytetrafluoroethylene (PTFE), ethylene-tetrafluoroethylene copolymer (ETFE), and tetrafluoroethylene- Examples thereof include hexafluoropropylene copolymer (FEP), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), polydimethylsiloxane (PDMS), and the like. The base material may be formed of a single material, and the cell placement portion and other portions (for example, wall portions) may be formed of different materials.

It is preferable that the cell placement part of the substrate has permeability to gas or liquid. When the cell placement part is permeable to gas or liquid, the cell placement part is made of a material selected from a porous material, a material having a mesh structure, and a material having a swelling property with respect to a liquid (preferably water). Preferably it is formed.
When cells mounting portion of the substrate has a permeability to gas, the permeability is, for example, 10 pmol/s/cm ² or more with respect to air, 600pmol / s / cm ² or more. In addition, when the cell placement part has swelling property with respect to the liquid or has a communicating hole, particularly excellent liquid permeability can be expected.

The base material is placed in the container, for example, in the container so that the surface of the cell placement portion and the bottom surface of the inner wall of the container are substantially parallel. In addition, the cell placement surface of the substrate is located in the liquid medium, and the distance from the bottom surface of the inner wall of the container to the surface of the liquid culture medium is from the bottom surface of the inner wall of the container to the surface of the cell placement portion of the substrate. More preferably, the base material is arranged such that the distance ratio is, for example, 0.1 or more and 0.95 or less, preferably 0.5 or more and 0.95 or less.

Cell sheet laminated body A cell sheet laminated body is mounted on the cell mounting part surface of a base material. As a method of placing the cell sheet laminate on the cell placement part surface of the base material, a cell sheet laminate prepared in advance may be placed on the cell placement part surface, and the cell is placed on the cell placement part surface. You may mount by laminating | stacking a sheet | seat and producing a cell sheet laminated body.

When the cell sheet laminate is prepared in advance, the cell sheet laminate is produced by laminating the cell sheets on the carrier substrate, and the cell sheet laminate is placed on the cell placement portion surface together with the carrier substrate. May be. The conveyance base material needs to be formed of at least a material having gas permeability, and more preferably formed of a material having gas permeability to a liquid in which the gas is dissolved. Specific examples of the substrate for conveyance include a porous film (hereinafter referred to as a VECELL film) constituting the bottom surface of VECELL Preset (trade name, manufactured by Bethel).

The cell sheet laminate can be prepared by laminating two or more cell sheets. Examples of the method for laminating cell sheets include a method of laminating two or more cell sheets in a liquid medium and then removing the liquid medium. Moreover, you may obtain a cell sheet laminated body by laminating | stacking a cell sheet laminated body instead of a cell sheet.

At least one inter-sheet fiber (filament) may be disposed between the cell sheets of the cell sheet laminate. For example, the inter-sheet fibers are arranged such that the length direction thereof is parallel to the surface of the cell sheet. Moreover, it is preferable that the at least one edge part of the fiber between sheets is located in the exterior of a cell sheet laminated body, ie, in a liquid culture medium. In another embodiment, at least one end of the inter-sheet fibers may be outside the liquid medium. By arranging the inter-sheet fibers between the cell sheets, the formation of the cultured cell structure proceeds more efficiently. About the detail of the fiber between sheets, it is the same as that of the fiber with which the base material mentioned later is provided.
As a method of arranging the inter-sheet fibers between the cell sheets, by placing the inter-sheet fibers on the cell sheet that is the lower layer when laminating the cell sheets, by laminating the cell sheet that is the upper layer on it, Can be placed between cell sheets.

When inter-sheet fibers are arranged between cell sheets, it is preferable to arrange one or more inter-sheet fibers per cell sheet, for example. Moreover, it can be 1 to 10 per 1 cm ^{2 of the} cell sheet area.

The number of cell sheets constituting the cell sheet laminate is, for example, 2 or more and 100 or less, and preferably 2 or more and 50 or less.
The thickness in the stacking direction of the cell sheet laminate is, for example, 20 μm or more, preferably 500 μm or more, for example, 10 mm or less, preferably 8 mm or less.

The area of the surface perpendicular to the stacking direction of the cell sheet laminate is, for example, 0.15 cm ² or more, preferably 7 cm ² or more, and 80 cm ² or less, preferably 10 cm ² or less.

The cell sheet constituting the cell sheet laminate is a sheet-like cell aggregate composed of one layer formed by adhering a plurality of cells. The cells constituting the cell sheet are not particularly limited and can be appropriately selected depending on the purpose and the like. The cells constituting the cell sheet are mammalian cells including humans, and examples include somatic cells, precursor cells thereof, and mixed cells thereof. Specific cells include endothelial cells, mesenchymal stem cells, adipose-derived stem cells, cord blood-derived stem cells, dental pulp stem cells, cardiomyocytes, heart wall cells, hepatocytes, fibroblasts, osteoblasts, vascular endothelial cells, liver Examples include progenitor cells, mesenchymal cells, islet cells, chondrocytes, epithelial cells, and the like, and at least one cell selected from the group consisting of these is preferred.

The cells constituting the cell sheet may be cells contained in a tissue isolated by an arbitrary method, or may be a cell line established from the tissue. In addition, cells derived from pluripotent stem cells by any method may be used. The guidance method used at this time can be a method well known to those skilled in the art, and is not particularly limited.

The cell sheet can be produced by culturing desired cells in a sheet form in a culture device and taking out the cells in a sheet state. As a method for taking out the cultured cells in a sheet state, for example, after culturing the cells using a culture device coated with a temperature-responsive polymer to form a cell sheet, the cell sheet is removed from the culture device by changing the temperature. The method of peeling can be mentioned.
Examples of the temperature-responsive polymer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives (see, for example, JP 2010-255001 A), or vinyl ether derivative polymers. Can be mentioned. As a preferable culture device, a culture device fixed with poly-N-isopropylacrylamide is exemplified. Such culture equipment can also be purchased from Cellseed as UpCell. In addition, as a culture device coated with a temperature-responsive coating agent, a culture device coated with methyl cellulose described in Chen CH, et al, Biomacromolecules. 7: 736-43, 2006, Takamoto Y, et al, A culture apparatus having a polyethylene terephthalate (PET) film coated with a block copolymer of 2-ethoxyethyl vinyl ether and 2-phenoxyethyl vinyl ether described in J. Biomater. Sci. Polymer Edn, 18: 1211-1222, 2007 Illustrated.

Culture of cell sheet laminate The cell sheet laminate placed on the cell placement part of the substrate is cultured in a liquid medium. By placing the cell sheet laminate on the substrate and culturing the cell sheet laminate close to the surface of the liquid medium, oxygen and the like are sufficiently supplied to the inside of the cell sheet laminate, and the cell culture is in good condition Done in Thereby, for example, cell adhesion and the like proceed between the cell sheets to form a cultured cell structure that can integrally exert a physiological function.

What is necessary is just to select desired culture conditions for the culture conditions of a cell sheet laminated body according to the cell type, cell number, etc. which comprise a cell sheet laminated body. The culture temperature is, for example, about 30 to 40 ° C, preferably about 37 ° C. The culture is performed, for example, in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is, for example, 2 to 5%. The culture time is, for example, 1 day to 10 days.

Culturing may be performed in a state where air is retained in the recess of the substrate. When the cell placement part of the base material is permeable to gas, by culturing while holding the air in the recess, the bottom surface of the cell sheet laminate is also adhered and cultured in the culture solution. The effect that it becomes possible to culture in a state close to the gas-liquid interface is obtained. At least a part of the air held in the recess may be exchanged between the start of culture and the end of culture. When exchanging the air held in the recess, the exchange amount can be appropriately selected according to the purpose or the like.

Further, as a method for exchanging air held in the recess, for example, a method of supplying / recovering air through a hollow gas exchange member having one opening in the recess can be cited. The air supplied to the recess may be released into the liquid medium from the cell mounting portion having gas permeability, and at least a part of the air may be recovered from the recess via the air exchange member.

The air retained in the recess may contain a component that gives a stimulus to the cultured cells. Examples of components that stimulate cultured cells include nitric oxide, nitrogen, carbon monoxide, carbon dioxide, hydrogen hydrogen sulfide, oxygen, and the like. When a component that stimulates cultured cells is included in the air held in the recess, the concentration can be appropriately selected according to the nature, purpose, and the like of the component.

Culturing may be performed in a state where the liquid medium is held in the recess of the base material. The liquid medium held in the recess may be the same as or different from the liquid medium stored in the container. When the cell placement part of the base material is permeable to liquid, by culturing while holding the liquid medium in the recess, the cell sheet laminate can also be cultured from the bottom of the cell sheet while adhering culture in the liquid medium. The effect that the supply of nutrients and oxygen through a liquid medium becomes possible is obtained. At least a part of the liquid medium held in the recess may be exchanged between the start of culture and the end of culture. When exchanging the liquid culture medium held in the recess, the exchange amount can be appropriately selected according to the purpose or the like.

In addition, as a method of exchanging the liquid medium held in the recess, for example, a method of supplying and recovering the liquid medium via a hollow liquid exchange member having one opening in the recess, or via a porous liquid exchange member And a method of exchanging the liquid medium. The liquid medium supplied to the recess through the hollow liquid exchange member may be discharged into the liquid medium from the cell mounting portion having liquid permeability, and at least a part of the liquid medium from the recess through the liquid exchange member. It may be recovered. When a porous liquid exchange member is used, the liquid medium may be exchanged by diffusion.

The liquid medium held in the recess may contain a component that stimulates the cultured cells. Examples of components that stimulate cultured cells include growth factors such as VEGF (Vascular endothelial growth factor) and bFGF (basic fibroblast growth factor), nitrogen monoxide, nitrogen, carbon monoxide, carbon dioxide, hydrogen, hydrogen sulfide, Examples thereof include oxygen. When the liquid medium held in the recess contains a component that stimulates cultured cells, the concentration can be appropriately selected according to the nature, purpose, etc. of the component.

The base material on which the cell sheet laminate is placed includes at least one fiber (filament) having one end penetrating into the cell placement portion and extending in the stacking direction of the cell sheet laminate. The fiber penetrates the cell placement portion toward the recess provided on the back surface of the cell placement portion. Moreover, the fiber extended in the lamination direction of a cell sheet laminated body may be in contact with the cell sheet laminated body, and may penetrate the cell sheet laminated body.
It is preferable that at least one of the fibers penetrates the cell sheet laminate. In particular, it is preferable that a plurality of fibers are arranged and most of the number of the fibers penetrates the cell sheet laminate. When the area of the cell sheet laminate is small, the number of non-penetrating fibers may be larger than the number of fibers that penetrate, and it consists only of fibers that do not penetrate the cell sheet laminate. May be.
By providing the substrate with fibers, for example, when the fibers have gas / liquid permeability, it is possible to supply a medium or air into the cell sheet laminate through the fibers, or the cell sheet laminate is placed. The effect that it is stably fixed to the part is obtained.

Examples of the fiber material include ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (FEP), CYTOP (trade name, manufactured by Asahi Glass Co., Ltd.), and Flemion (trade name, Asahi Glass). Teflon AF1600 (trade name, manufactured by Mitsui DuPont Fluorochemical Co., Ltd.), Teflon AF2400 (trade name, manufactured by Mitsui DuPont Fluorochemical Co., Ltd.), polydimethylsiloxane (PDMS), cross-linked gelatin hydrogel, etc. It is preferable to include an oxygen permeable material. Furthermore, it is also preferable that the material is capable of supplying a medium. Moreover, it is preferable that a fiber swells with a culture medium. When it swells, the supply of oxygen is improved along with the medium.

The fiber structure may be any of a porous body, a hollow body, a solid body, and the like. From the viewpoint of exchanging liquid or gas with the concave portion, a porous body or a hollow body is preferable. When the fiber is a porous body or a hollow body, the liquid or gas can be easily exchanged. That is, the fiber provided in the base material may serve as the gas exchange member or the liquid exchange member.

The thickness of the fiber is, for example, 0.1 μm or more and 5000 μm or less as a diameter, preferably 1 μm or more and 1000 μm or less, and more preferably 10 μm or more and 500 μm or less. When the fiber is a hollow body, the diameter of the hollow portion is, for example, not less than 0.1 μm and not more than 5000 μm, preferably not less than 10 μm and not more than 500 μm.
The length of a fiber will not be specifically limited if it is longer than the thickness of a cell sheet laminated body, For example, they are 2 mm or more and 50 mm or less, Preferably they are 5 mm or more and 30 mm or less.

What is necessary is just to select suitably the number of the fibers with which a base material is provided according to the thickness of a cell sheet laminated body, a magnitude | size, etc. The number of fibers is, for example, from 0.01 to 1000, preferably from 0.1 to 100, and more preferably from 0.1 to 50, per 1 cm ^{2 of the} cell sheet area.
The ratio of the number of fibers penetrating the cell sheet laminate to the total number of fibers provided in the substrate is preferably 50% to 100%, more preferably 80% to 100%.

A method for culturing a cell sheet laminate on a substrate having fibers will be described with reference to the drawings. FIG. 1 is a schematic cross-sectional view schematically showing an example of a method for culturing a cell sheet laminate according to the present embodiment. In FIG. 1, the liquid medium 16 is accommodated in the container 2, and the base material 4 is disposed inside the container 2. A cell sheet laminate 14 is placed on the surface of the cell placement portion 6 of the base 4 via a transport base 12. In another aspect, the cell sheet laminated body 14 may be directly mounted on the surface of the cell mounting part 6 without using the conveyance base material 12. A recess 8 is formed on the back surface of the cell placement unit 6, and a liquid medium 16 is held in the recess 8. Air may be held in the recess 8 instead of the liquid medium 16. The cell sheet laminate placed on the surface of the cell placement unit 6 is cultured at a position closer to the surface of the liquid medium than placed on the bottom surface 18 of the inner wall of the container.

Moreover, the base material 4 is provided with the fiber 10 which penetrates the cell mounting part 6 toward the recessed part 8, and the fiber 10 penetrates the cell sheet laminated body 14, and is extended in the lamination direction of the cell sheet laminated body 14. Yes. In FIG. 1, the fiber 10 penetrates the cell sheet laminate 14 in the lamination direction, but may contact the side surface of the cell sheet laminate 14 without penetrating the cell sheet laminate 14. In FIG. 1, one end of the fiber 10 is located in the recess 8 and the other end is located in the liquid medium, but the other end may be located outside the liquid medium 16. .

The cultured cell structure produced by the production method of the present embodiment has a feature of maintaining high cell activity while having a thickness that causes necrosis inside the conventional culture method, such as drug discovery research and regenerative medicine. It can be applied to any use.

Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples. The cultured cell structure was evaluated by immunostaining and Western blot analysis as described below.

Immunostaining The obtained cultured cell structure was washed twice with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde, solidified with OCT compound, sliced into 10 μm thickness with a cryostat, and frozen section. Was made. The frozen section was prepared so that a cross section parallel to the stacking direction could be observed.
Immunostaining includes hypoxia probe (Hypoxyprobe), a hypoxia marker, pyruvate dehydrogenase (PDH) antibody (SANTA CRUZ BIOTECHNOLOGY), a cell respiration marker, Ki-67 antibody (Santa TA), a cell proliferation marker. BIOTECHNOLOGY). Images of immunostained cultured cell structures are shown in FIGS.

Western blot analysis The obtained cultured cell structure was placed in a Lysis buffer and homogenized, and the supernatant was collected. After diluting with sodium dodecyl sulfate (SDS) sample buffer, it was applied to an acrylamide gel to 50 μg / well, and the protein was developed by electrophoresis. The developed protein was transferred to a PVdF (Polyvinylidene difluoride) membrane and treated with primary antibodies of pyruvate dehydrogenase and Ki-67, respectively. After treatment for a predetermined time, the PVdF membrane was washed with TBST (Tris Buffered Saline with Tween 20), and then treated with an HRP (horseradish peroxidase) -conjugated secondary antibody. After the treatment for a predetermined time, it was washed again with TBST, treated with SuperSignal Chemiluminescent Substrate (Thermo scientific), and visualized using LAS4000 (trade name, manufactured by Fuji Film). The obtained image was subjected to relative quantification using image analysis software Image J.
The evaluation results are shown in FIGS.

(Reference example)
Production of Hollow Fiber A resin was coated on the outer surface of a syringe needle (Terumo) having an inner diameter of 400 μm using a dip coater. A coating film was prepared by adjusting the solution concentration and the pulling speed so that the resin film thickness in a dry state was 20 to 30 μm. Note that a sacrificial layer may be precoated on the syringe needle in order to improve the peelability from the needle.
Preparation of Cell Sheet A liquid medium prepared by adding 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin to MEMα was prepared. A cell culture polystyrene 6-well plate is seeded with 2 mL of a medium in a human mesenchymal stem cell line (hMSC) to 200,000 cells / well, and cultured for 8 to 10 days while changing the medium every 3 days. A cell sheet was formed on the plate.

Preparation of cell sheet laminate A 10 cm cell culture PS petri dish was treated with a 0.1% gelatin aqueous solution at room temperature for 10 minutes. The cell sheet formed as described above was peeled off by pipetting, and the peeled cell sheet was sucked with a decantation or pipette tip having a large diameter and transferred to a gelatin-coated PS petri dish. The medium was dripped with a pipette to stretch the cell sheet wrinkles. The dropped medium was sucked off and allowed to stand in an incubator at 37 ° C. and 5% CO ₂ for 30 minutes to adhere the cell sheet to the petri dish.
In the same manner, the next cell sheet is transferred to a petri dish together with the culture medium, stacked on the adhered cell sheet, and after dropping the culture medium with a pipette to stretch the cell sheet wrinkles, the culture medium is sucked off, and the medium is sucked at 37 ° C, 5% CO _2. In the incubator for 30 minutes to laminate the cell sheets.
The cell sheet lamination operation was repeated a predetermined number of times to obtain a cell sheet laminate in which a predetermined number of cell sheets were laminated. When the laminate was transferred to the cell placement section and cultured, a VECELL film was used as a transport substrate, and the laminate peeled off from the PS petri dish with a spatula or the like was adsorbed to the VECELL film and transferred to the placement section. When culturing in a PS petri dish, the medium was poured directly into the laminate and cultured. In addition, when stacking 6 or more cell sheets, the stacked body in which 3 to 10 sheets are stacked is transported onto another stacked body using a VECELL film, and the stacked body is peeled off. It can also be produced by sequentially laminating each other.

(Comparative Example 1)
A cell sheet laminate comprising 5 cell sheets was placed on the bottom of the inner wall of a 10 cm cell culture PS petri dish, and 10 mL of MEM-α medium (10% fetal bovine serum, 1% penicillin / streptomycin) was added. Culturing was performed for a predetermined period in an incubator at 37 ° C. and 5% CO ₂ .
The cultured cell structure obtained after the culture was evaluated by immunostaining by the frozen section method and Western blot analysis.
Images obtained by immunostaining the hypoxia marker, cell proliferation marker and cell respiration marker are shown in FIGS. 6, 8 and 10, respectively. The results of Western blot analysis are shown in FIGS.

(Comparative Example 2)
The circumference of the LUMOX 35 mm dish was cut to adjust the height. The dish was inverted in a 10 cm cell culture PS petri dish, adhered so that the bottom surface of the dish became a cell placement portion, and sterilized to obtain a culture substrate. A cell sheet laminate comprising 10 cell sheets formed on the VECELL film was placed on the bottom of the dish, and the medium was added to such an extent that the outermost surface of the cell sheet laminate was covered. At this time, the ratio of the height of the bottom surface of the dish to the height of the medium in the petri dish was 0.9. The cells were cultured for a predetermined period in an incubator at 37 ° C. and 5% CO ₂ and evaluated by Western blot analysis. The results of Western blot analysis are shown in FIG.

Example 1
CYTOP CTL-109S (trade name, manufactured by Asahi Glass Co., Ltd.) was coated on the outer surface of a syringe needle having an inner diameter of 400 μm to produce a hollow filament having a wall thickness of about 30 μm. The SEM image of the produced hollow filament is shown in FIG. The left figure of FIG. 12 is an image with a magnification of 100 times, and the right figure is an image with a magnification of 1000 times.
After placing the cell sheet laminate consisting of five cell sheets formed on the VECELL film on the bottom of the dish, the hollow filament is penetrated through the cell sheet laminate, the VECELL membrane and the dish bottom together with the syringe needle, and the filament The syringe needle was pulled out while pressing with tweezers. The remaining filament was in a state where one end was in the dish and the other end was exposed to the air from the medium surface. The cell sheet was the same as Comparative Example 2 except that the filaments were arranged in a state of penetrating the cell sheet laminate at a rate of ² per 0.8 cm ² and that the number of laminated cell sheets was 5. Laminates were cultured and evaluated by immunostaining by Western section and Western blot analysis. The evaluation results are shown in FIGS. Moreover, the image which immunostained the hypoxia marker, the cell proliferation marker, and the cell respiration marker is shown in FIG.5, FIG.7 and FIG.9, respectively.

From the result of immunostaining, the staining of hypoxic marker molecule (Hyoxyprobe) was weaker in Example 1 than in Comparative Example 1 (FIGS. 6, 8 and 10), and the cell respiration marker protein (PDH) and the cells Staining of proliferation marker protein (Ki-67) was strongly observed.
Also, from the results of Western blot analysis (FIGS. 2 and 3), the expression level of cell respiration marker protein (PDH) and cell proliferation marker protein (Ki-67) was higher in Example 1 than in Comparative Example 1. I understand that.

(Example 2)
The cell sheet laminate was cultured and evaluated in the same manner as in Example 1 except that Teflon AF1600 (trade name, manufactured by Mitsui DuPont Fluorochemical Co., Ltd.) was used instead of CYTOP CTL-109S. Results similar to 1 were obtained.

(Example 3)
CYTOP CTL-109S was coated on the outer surface of a syringe needle having an inner diameter of 400 μm to produce a hollow filament. After placing the cell sheet laminate formed on the VECELL membrane on the bottom of the dish, the hollow filament is passed through the cell sheet laminate, the VECELL membrane and the bottom of the dish together with the syringe needle, and the syringe needle while pressing the filament with tweezers Pulled out. The remaining filament was in a state where one end was in the dish and the other end was exposed to the air from the medium surface. The cell sheet laminate was cultured in the same manner as in Example 1 except that only one filament was disposed so as to penetrate the center of the cell sheet laminate. After culturing for 4 days, the cell sheet laminate was punched out to 5 mmφ centering on the position where the fiber penetrated, and the expression level of PDH as a cell respiration marker was evaluated in the same manner as in Example 1. The evaluation results are shown in FIG. The vertical axis in FIG. 4 is the PDH expression level corrected with the actin expression level.

(Comparative Example 3)
The cell sheet laminate was cultured and evaluated in the same manner as in Example 3 except that only one 400 μm diameter syringe needle was used instead of the filament so as to penetrate the center of the cell sheet laminate. The evaluation results are shown in FIG.

FIG. 4 shows that the amount of PDH expressed was higher in Example 3 using fibers than in Comparative Example 3 using syringe needles. In the one-sided test, a significant difference was recognized at a significance level of 5%.

Example 4
When the cell sheet laminate was cultured and evaluated in the same manner as in Example 3 except that Teflon AF1600 was used instead of CYTOP CTL-109S, the same results as in Example 3 were obtained.

(Example 5)
Flemion EW909 (trade name, manufactured by Asahi Glass Co., Ltd.) was used instead of CYTOP CTL-109S, a gelatin film was pre-coated on the syringe needle as a sacrificial layer, and Flemion EW909 was annealed in an oven at 120 ° C. for 10 minutes, 1.76 cm ² The cell sheet laminate was cultured and evaluated by Western blot analysis in the same manner as in Comparative Example 2 except that the filaments were arranged in a state of penetrating the cell sheet laminate at a rate of about 1. The wall thickness of the produced hollow filament was about 25 μm. FIG. 13 shows an SEM image of the produced hollow filament. The left figure of FIG. 13 is an image with a magnification of 100 times, and the right figure is an image with a magnification of 1000 times.
The evaluation results are shown in FIG.

(Example 6)
Flemion EW909 was used in place of CYTOP CTL-109S, a gelatin film was precoated as a sacrificial layer on the outer surface of the syringe needle, and Flemion was annealed in an oven at 120 ° C. for 10 minutes at a rate of 3 per 1.76 cm ^2. The cell sheet laminate was cultured and evaluated by Western blot analysis in the same manner as in Comparative Example 2 except that the filament was disposed in a state of penetrating the cell sheet laminate. The evaluation results are shown in FIG.
It can be seen from FIG. 11 that Comparative Example 2 using no fiber showed significantly higher PDH expression in Examples 5 and 6 using fiber. Moreover, the tendency for the PDH expression level to become higher is seen when the number of fibers is three than one.

(Example 7)
A hollow filament was prepared by coating gelatin hydrogel on the outer surface of a syringe needle having an inner diameter of 400 μm and thermally crosslinking in a vacuum oven at 140 ° C. for 72 hours. The cell sheet laminate was cultured and evaluated in the same manner as in Example 3 except that a crosslinked gelatin hydrogel was used instead of CYTOP CTL-109S, and the same results as in Example 3 were obtained. .

(Example 8)
Flemion EW909 was used instead of CYTOP CTL-109S, a gelatin film was precoated as a sacrificial layer on the outer surface of the syringe needle, Flemion EW909 was annealed in an oven at 120 ° C. for 10 minutes, and between the cell sheets. A cell sheet laminate was cultured in the same manner as in Example 3 except that 45 sheets were laminated by sandwiching gelatin hydrogel particles having a particle diameter of 20 μm to 32 μm by the method described in 1.
After culturing for a predetermined period and examining whether the cell sheet laminate was stably held on the substrate, the laminate was stably held on the substrate even after culturing for 2 days or more.

(Comparative Example 4)
A cell sheet laminate in which gelatin hydrogel particles having a particle diameter of 20 μm to 32 μm were sandwiched between the cell sheets by the method described in Patent Document 2 and 45 cell sheets were stacked was not disposed in the dish. The cell sheet laminate was cultured in the same manner as in Example 1 except that it was placed on the bottom of the inner wall, and it was examined whether the cell sheet laminate was stably cultured on a petri dish.
As a result, on the second day of culture, the cell sheet laminate peeled from the petri dish and contracted, or peeled between the sheets and fell apart.
It should be noted that the entire content of the specification, claims, drawings and abstract of Japanese Patent Application No. 2016-200864 filed on October 12, 2016 is cited herein as the disclosure of the specification of the present invention. Incorporated.

2: Container, 4: Substrate, 6: Cell placement unit, 8: Recess, 10: Fiber, 12: Transport substrate, 14: Cell sheet laminate, 16: Liquid medium

Claims (15)

Placing a cell sheet laminate in which two or more cell sheets are laminated on the surface of a cell placement part of a substrate placed in a container containing a liquid medium;
Culturing the cell sheet laminate on the cell placement part of the substrate in a liquid medium;
Including
The base material has a recess on the back surface of the cell placement portion,
The base material includes at least one fiber penetrating the cell placement portion toward the concave portion, and the cell sheet laminate is cultured in a state where the fiber extends in the stacking direction. Manufacturing method. The manufacturing method according to claim 1, wherein at least one of the fibers penetrates the cell sheet laminate. The ratio of the distance from the inner wall bottom surface of the container to the cell placement surface of the substrate to the distance from the inner wall bottom surface of the container to the surface of the liquid medium is 0.1 or more and 0.95 or less. 3. The production method according to 1 or 2. The production method according to any one of claims 1 to 3, wherein the cell placement portion of the substrate has permeability to a gas or a liquid. The production method according to any one of claims 1 to 4, wherein the culture is performed with air held in the recess. The production method according to any one of claims 1 to 4, wherein the culture is performed with a liquid medium held in the recess. The production method according to any one of claims 1 to 6, wherein an end of the fiber arranged on the surface side of the cell placement unit is located in a liquid medium. The production method according to any one of claims 1 to 6, wherein an end of the fiber arranged on the surface side of the cell placement unit is located outside the liquid medium. The production method according to any one of claims 1 to 8, wherein the fiber is a porous body. The production method according to any one of claims 1 to 8, wherein the fiber is a hollow body. The manufacturing method according to any one of claims 1 to 10, wherein the fiber includes an oxygen-permeable material. The cell sheet is at least selected from the group consisting of endothelial cells, mesenchymal stem cells, adipose-derived stem cells, umbilical cord blood-derived stem cells, dental pulp stem cells, cardiomyocytes, heart wall cells, hepatocytes, fibroblasts and osteoblasts. The production method according to any one of claims 1 to 11, which is a cell sheet derived from one type of cell. The production method according to any one of claims 1 to 12, wherein the cell sheet laminate has 2 or more and 100 or less cell sheets. The production method according to any one of claims 1 to 13, wherein the cell sheet laminate has inter-sheet fibers between cell sheets. The manufacturing method according to claim 14, wherein at least one end of the inter-sheet fiber is located in a liquid medium.

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