JP2021058175A - Cell presser, cell layer preparation tool, and cell layer production method - Google Patents

Abstract

To provide a technology for producing a cell tissue having a constant thickness, which can be evaluated and observed as a single cell.SOLUTION: A cell presser 1A comprises a cell-pressing member 11 having a cell non-adhesive region and inserted into a recess 3 of a culture vessel 2; and a support mechanism 12 that supports the cell-pressing member 11 inserted into the recess 3 so that a pressing surface 11a at the lower end of the member is arranged in parallel to an inner bottom surface 3a of the recess 3 at a position separated upward from the inner bottom surface 3a of the recess 3.SELECTED DRAWING: Figure 3

Description

The present invention relates to a cell retainer, a cell layer preparation tool, and a cell layer production method.

When cells are cultured to prepare a monolayer or multi-layer cell tissue, the cells may aggregate or spheroids may occur on a part of the culture surface, the peripheral edge of the incubator, or the like. Therefore, it is difficult to prepare a cell tissue having a constant thickness and a flat surface. In particular, when cells aggregate or spheroids occur at the marginal portion, there is a difference in the refractive index between the marginal portion and other parts, which causes a problem in observing the cell tissue.

So far, as a method for producing a flat cell tissue having a constant thickness, a method for producing a cell sheet having a certain thickness or more by stacking cell sheets has been known.

For example, in Patent Document 1, for the purpose of efficiently producing a laminated cell sheet composition in a short time, cell sheets are laminated by applying a cell adhesive substance to the layered cell sheets and applying pressure. A method of reducing the time required for this is disclosed.

However, in the method of laminating cell sheets, an adhesive substance is used when laminating the cell sheets. In particular, when thrombin and fibrinogen are used, there is a problem that fibrin gel is formed and it is not possible to evaluate or observe only cells.

Therefore, an object of the present invention is to provide a technique for producing a cell tissue body in which the thickness of the cell tissue is constant and the cell itself can be evaluated and observed.

The present invention includes the following aspects.
The cell pressing member having a non-cell adhesive region and inserted into the recess of the culture vessel and the cell pressing member inserted into the recess are separated from each other by the pressing surface at the lower end thereof from the inner bottom surface of the recess. A cell retainer having a support mechanism that supports the recess so as to be arranged parallel to the inner bottom surface of the recess.

According to the present invention, the present invention can provide a technique for producing a cell tissue composed of only flat cells having a constant thickness while controlling the thickness of the tissue without using an adhesive substance. ..

It is a partial right sectional view of the cell holding tool 10. It is a top view of the cell retainer 10. It is a partial normal sectional view of the cell layer preparation tool 1A. It is a partial normal sectional view of the cell layer preparation tool 1A. It is a partial normal sectional view of the cell layer preparation tool 1A. It is a partial normal sectional view of the cell layer preparation tool 1B. It is a partial normal sectional view of the cell layer preparation tool 1C. It is a partial normal sectional view of the cell layer preparation tool 1D. It is a partial right sectional view of the cell layer preparation tool 1E. It is a partial normal sectional view of the cell layer preparation tool 1F. It is a top view of the cell layer preparation tool 1F. It is a partial normal sectional view of the cell layer preparation tool 1A. It is a partial normal sectional view of the cell layer preparation tool 1A. It is a partial normal sectional view of the cell layer preparation tool 1A. It is a partial normal sectional view of the cell layer preparation tool 1A.

[Cell holder]
The cell retainer and the cell layer preparation tool according to one embodiment of the present invention will be described with reference to the drawings. In addition, in FIGS. 1, 3 to 10, and 12 to 15, the upper side is referred to as the upper side and the lower side is referred to as the lower side.

As illustrated in FIG. 3 and the like, the cell layer preparation tool 1A is composed of a cell retainer 10A and a culture vessel 2.

The culture vessel 2 illustrated in FIG. 3 has a recess 3 formed from the upper surface 4 thereof. The inner bottom surface 3a of the recess 3 is a flat surface extending perpendicularly to the depth direction of the recess 3.

Examples of the culture container 2 include, but are not limited to, bottles, flasks, dishes, multi-dish, well plates, and the like, and culture containers known to those skilled in the art can be used.

The inner bottom surface 3a of the recess 3 of the culture vessel 2 is provided with cell adhesion. As a method for imparting cell adhesion, known methods such as plasma treatment and addition of cell adhesive material can be used.

As shown in FIGS. 1 and 3, the cell presser 10A has a cell presser member 11 inserted into the recess 3 of the culture vessel 2 and a pin-shaped protrusion protruding from the presser surface 11a at the lower end of the cell presser 10A. (Support mechanism) 12 is provided. Further, the cell presser 10A also includes a spacer projection 13 projecting from the side surface 11b of the cell presser member 11.

The cell holding member 11 illustrated in FIGS. 1 to 5 is formed in a disk shape. The pressing surface 11a of the cell pressing member 11 is one surface of the plate-shaped (disk-shaped in FIGS. 1 to 5) cell pressing member 11.
The cell holding member 11 is not limited to a disk shape, and may have a polygonal plate shape (polygonal plate shape) such as a quadrangle, a pentagon, or a hexagon on both sides. Further, the cell holding member 11 is not limited to a plate shape in which the maximum dimension from the central axis passing through the centers of both sides thereof to the side surface is larger than the thickness, and the columnar shape having a thickness larger than the maximum dimension from the central axis to the side surface. It may be. The columnar cell holding member 11 may include a polygonal columnar shape extending in a polygonal cross-sectional shape such as a quadrangle, a pentagon, or a hexagon, or a columnar column extending in a circular cross section. Further, the columnar cell pressing member 11 uses the end surface on one side in the central axis direction as the pressing surface 11a.

The pressing surface 11a of the cell pressing member 11 is a flat surface. Since the pressing surface 11a is a flat surface, a flat cell layer 6 can be produced.

As shown in FIGS. 1 and 3, a plurality of pin-shaped protrusions 12 project vertically from the holding surface 11a of the cell holding member 11, and the length from the tip of each pin-shaped protrusion 12 to the holding surface 11a is It is the same.

The protrusion 12 may be integrated with the holding member 11 of the cell holding tool 10A, or may be a separate body. As the separate protrusion 12, for example, one that is partially embedded and fixed to the cell holding member 11 or mechanically fixed by screwing or the like can be adopted.

The surface layers of the cell holding member 11, the protrusion 12, and the spacer protrusion 13 may be formed of a non-cell adhesive material. Examples of the cell non-adhesive material of the cell holding member include, but are not limited to, glass and PDMS (Poly Dimethicylpolysiloxane), and a cell non-adhesive material known to those skilled in the art can be used. ..

The surfaces of the cell holding member 11, the protrusion 12, and the spacer protrusion 13 may be subjected to cell non-adhesive treatment. The cell non-adhesive treatment may be, for example, applying 2-methacryloyloxyethyl phosphorylcholine (MPC polymer), but is not limited thereto. Regarding the range of the cell non-adhesive treatment of the cell pressing member 11, at least the region of the pressing surface 11a and the side surface 11b in contact with the pressing surface 11a that comes into contact with or may come into contact with the cells is not cell. It may be an adhesive material or may be treated with a non-cell adhesive treatment. By doing so, it is possible to suppress the influence on the cells when the cell holding member 11 is separated from the cells.

As illustrated in FIG. 2, the spacer protrusions 13 project from the side surface 11b of the cell pressing member 11 and are dispersedly arranged at a plurality of locations in the circumferential direction of the side surface of the cell pressing member 11. In FIG. 2, the plurality of spacer protrusions 13 are evenly arranged at intervals in the circumferential direction of the side surface of the cell holding member 11.

As illustrated in FIG. 4, the spacer protrusions 13 include a horizontal bar 13b protruding from the side surface of the cell holding member 11 and a vertical bar 13a connected to the tip of the horizontal bar 13b and extending in a direction perpendicular to the holding surface 11a. have.
In FIG. 2, the horizontal bar 13b of the spacer projection 13 projects from the side surface of the cell pressing member 11 in the direction along the pressing surface 11a.
The vertical bar 13a of the spacer protrusion 13 has a portion extending downward from the horizontal bar 13b (hereinafter, also referred to as a lower extending portion) and a portion extending upward from the horizontal bar 13b (hereinafter, extending upward). It also has a part). The downward protrusion dimension of the cell pressing member 11 of the lower extending portion of the vertical bar 13a from the pressing surface 11a is the same as the downward protrusion dimension of the cell pressing member 11 of the plurality of pin-shaped protrusions 12 from the pressing surface 11a. It is arranged in.

As illustrated in FIG. 4 and the like, the tip of the protrusion 12 protruding from the pressing surface 11a is abutted against the inner bottom surface 3a of the recess 3. Further, as illustrated in FIG. 4, the tip (lower end) of the lower extending portion of the horizontal bar 13b of the vertical bar 13a abuts on the inner bottom surface 3a of the recess 3 from above.
In the spacer projection 13 illustrated in FIG. 4 and the like, the tip of the lower extending portion of the vertical bar 13a abuts on the inner bottom surface 3a of the recess 3, so that the cell pressing member 11 is pressed and the pressing surface 11a is inside the recess 3. It serves as a protrusion that supports the recess 3 so as to be parallel to the inner bottom surface 3a at a position separated upward from the bottom surface 3a.

As illustrated in FIG. 4, the horizontal bar 13b is in contact with the inner peripheral surface 3b of the recess 3, and the vertical bar 13a is the cell pressing member 11 inserted into the recess 3 and the entire circumference of the inner peripheral surface 3b of the recess 3. A clearance of 18 is secured between them.

The spacer protrusion 13 also serves as a protrusion 12 that protrudes from the cell holding member 11 and comes into contact with the inner bottom surface 3a of the recess 3 from above. As described above, the protruding dimension of the cell pressing member 11 of the spacer projection 13 downward from the pressing surface 11a and the length of the plurality of pin-shaped protrusions 12 are the same. Therefore, when the spacer protrusion 13 abuts on the inner bottom surface 3a of the recess 3, the pressing surface 11a of the cell pressing member 11 is parallel to the inner bottom surface 3a of the recess 3 and is separated upward from the inner bottom surface 3a of the recess 3. It plays a role of supporting the position.

Next, an example of a cell layer manufacturing method for manufacturing a cell layer using the cell layer preparing tool 1A will be described.
First, as illustrated in FIG. 3, cells are put into the recess 3 and adhered to the inner bottom surface 3a of the recess 3 which has undergone cell adhesion treatment. Further, cells are put into the recess 3, and cells are deposited thicker than the protrusion dimension from the holding surface 11a of the cell holding member 11 of the protrusion 12 to form a cell deposition layer 5, and the culture solution is put into the recess 3. The cells of the cell deposition layer 5 are not closely adhered to each other, and the cell deposition layer 5 has a fluidity that easily flows with respect to an external force such as a pressing force.

Subsequently, as illustrated in FIG. 4, the cell holding member 11 of the cell holding tool 10A is inserted into the recess 3 from above. The cell holding member 11 is inserted into the recess 3 until the tip of the protrusion 12 abuts on the inner bottom surface 3a of the recess 3. As a result, the cell pressing member 11 is supported at a position separated upward by the size of the protrusion 12 from the inner bottom surface 3a of the recess 3.

The pressing surface 11a of the cell pressing member 11 inserted into the recess 3 has the tip of the protrusion 12 on the inner bottom surface 3a of the recess 3 in the process of lowering the cell pressing member 11 with respect to the recess 3 toward the inner bottom surface 3a. Pressing of the cell deposition layer 5 is initiated before bumping. The deposited cell deposition layer 5 before culturing has high fluidity, and when pressed by the cell pressing member 11, it is easily deformed and comes into surface contact with the pressing surface 11a of the cell pressing member 11.

When the cell deposit layer 5 is pressed by the cell retainer member 11, some of the cells forming the cell deposit layer 5 have a clearance between the cell retainer member 11 and the entire circumference of the inner peripheral surface 3b of the recess 3 by the spacer projection 13. It flows out to 18. The outflowed cells are removed by suction with a pipette or the like, and the edge portion of the cell deposition layer 5 located outside the pressing surface 11a of the cell pressing member 11 in the surface direction is flattened. The suction removal of cells in the clearance 18 with a pipette is performed by inserting the tip of the pipette into the clearance 18. The marginal portion of the cell deposition layer 5 is flattened flush with the pressing surface 11a.
For example, the fluidity of the cell deposition layer 5 is very high, and by adjusting the amount of cells input into the recess 3, the fluidity of the cell deposition layer 5 causes the cell deposition layer 5 to have a pressing surface 11a of the cell pressing member 11. When a flat upper surface is obtained, the suction of cells in the clearance 18 by the pipette can be omitted.

Subsequently, the cells forming the cell deposition layer 5 are cultured while maintaining the abutting state of the recess 3 at the tip of the protrusion 12 against the inner bottom surface 3a, and the cells are adhered to each other. That is, the cells of the cell deposition layer 5 are cultured and adhered to each other while maintaining the distance from the inner bottom surface 3a of the recess 3 of the pressing surface 11a of the cell pressing member 11 to form the cell layer 6 (see FIG. 5). .. The cell layer 6 is formed with an upper surface corresponding to the pressing surface 11a of the cell pressing member 11. Since the pressing surface 11a of the cell pressing member 11 is a flat surface parallel to the inner bottom surface 3a of the recess 3, a cell layer 6 having a flat upper surface corresponding to the pressing surface 11a of the cell pressing member 11 is formed in the recess 3. it can.
The cells in the cell deposition layer 5 are cultured for a preset time length of culture time. The culturing time is, for example, 2 to 3 hours, but is not limited to this.

After culturing the cells in the cell deposition layer 5 at the culture time of the preset time length is completed, the cell retainer 10A is then moved upward and taken out from the recess 3 as illustrated in FIG. By moving it above the recess 3, a cell layer 6 having a constant thickness and a flat upper surface remains in the recess 3. Since the pressing surface 11a of the cell pressing member 11 is parallel to the inner bottom surface 3a of the recess 3, the upper surface and the bottom surface of the cell layer 6 have parallel surfaces. Since the upper surface of the cell layer 6 is flat, it is suitable for observation with a microscope or the like.

In FIG. 5, the vertical bar 13a of the spacer protrusion 13 has a length protruding upward from the recess 3.
In FIG. 5, the portion of the vertical bar 13a of the spacer protrusion 13 that protrudes upward from the recess 3 serves as a handle that is gripped by fingers or the like in order to remove the cell retainer 10A from the recess 3.

FIG. 6 shows a modified example of the cell retainer 10B.
In addition, FIG. 6 also shows a cell layer preparation tool 1B composed of the cell retainer 10B and the culture vessel 2 of the modified example. The cell layer preparation tool 1B is obtained by changing the cell presser 10A to the cell presser 10B of FIG. 6 with respect to the cell layer preparation tool 1A of FIGS. 3 to 5.

In the cell presser 10B illustrated in FIG. 6, the protrusion 12 protruding from the presser surface 11a of the cell presser member 11 is omitted from the cell presser 10A described with reference to FIGS. 1 to 5, and the cell presser member 11 has a protrusion 12 thereof. A protrusion 14 (hereinafter, also referred to as a side protrusion) protruding from the side surface 11b is provided.
The side protrusions 14 are evenly arranged at a plurality of positions separated from each other in the circumferential direction of the side surface 11b of the cell holding member 11.

The lateral protrusion 14 of the cell layer preparation tool 1B illustrated in FIG. 6 hits the upper surface 4 of the culture vessel 2 from above when the cell holding member 11 is inserted into the recess 3 of the culture vessel 2 from above. Contact. In the cell holding member 11 of the cell layer preparation tool 1B illustrated in FIG. 6, the side protrusion 14 abuts on the upper surface 4 of the culture vessel 2, so that the inner bottom surface 3a of the recess 3 is in the depth direction with respect to the recess 3. It is positioned at a position separated upward from. Further, in the cell holding member 11 of the cell layer preparation tool 1B illustrated in FIG. 6, each side protrusion 14 around the cell holding member 11 abuts on the upper surface 4 of the culture container 2, so that the side protrusion 14 makes the culture container 2 relative to the cell holding member 11. The pressing surface 11a is supported so as to be arranged parallel to the inner bottom surface 3a at a position separated upward from the inner bottom surface 3a of the recess 3.
The side protrusion 14 serves as a support mechanism for supporting the cell pressing member 11 so that the pressing surface 11a is arranged parallel to the inner bottom surface 3a at a position separated upward from the inner bottom surface 3a of the recess 3.

In the cell layer preparation tool 1B illustrated in FIG. 6, after putting the cell deposition layer 5 and the culture solution in the recess 3 as shown in FIG. 3, the cell holding member 11 is inserted into the recess 3 and the side protrusion 14 is formed. By abutting the upper surface 4 of the culture vessel 2, the cell deposition layer 5 can be brought into surface contact with the pressing surface 11a of the cell pressing member 11. The cell layer preparation tool 1B illustrated in FIG. 6 cultivates the cell deposition layer 5 in surface contact with the pressing surface 11a of the cell pressing member 11 while maintaining the contact of the side protrusions 14 with the upper surface 4 of the culture vessel 2. After forming the cell layer 6, it can be removed from the recess 3 simply by moving it upward with respect to the culture vessel 2. The cell layer preparation tool 1B illustrated in FIG. 6 can be used for forming (preparing) a cell layer 6 having a flat upper surface.

In the cell layer preparation tool 1B illustrated in FIG. 6, a pipette is inserted between the side protrusions 14 which are in contact with the upper surface 4 of the culture vessel 2, and the side surface 11b of the cell holding member 11 and the inner peripheral surface 3b of the recess 3 are formed. A configuration in which a gap for inserting up to the clearance 18 secured between the two is secured can be preferably adopted.
However, the cell layer preparation tool 1B illustrated in FIG. 6 can also adopt a configuration in which a gap into which a pipette can be inserted is not secured between the side protrusions 14 in contact with the upper surface 4 of the culture vessel 2.

The cell layer preparation tool 1C illustrated in FIG. 7 includes a culture container 2 having a plurality of recesses 3 and a lid 15 for opening and closing the opening of the recess 3 of the culture container 2. The lid 15 includes a lid plate 15a and a plurality of cell retainers 10A provided on one side of the lid plate 15a. The cell holding tool 10A includes a plurality of protrusions 12 (supporting mechanisms) protruding from the holding surface 11a of the cell holding member 11.

On the upper surface 4 of the culture vessel 2 of the cell layer preparation tool 1C illustrated in FIG. 7, a sealing material 4a formed of a highly flexible polymer material such as rubber or elastomer is provided. The lid plate 15a of the lid 15 comes into contact with the sealing material 4a of the culture vessel 2 from above when the cell holding member 11 is inserted into the recess 3 of the culture vessel 2 from above. The cell holding members 11 of the plurality of cell holding tools 10A of the lid 15 are simultaneously inserted into the plurality of recesses 3 of the culture container 2 by abutting the lid plate 15a against the upper surface 4 of the culture container 2.

The cell pressing member 11 inserted into the recess 3 of the culture vessel 2 has a pressing surface due to the plurality of protrusions 12 by abutting the tip of the protrusion 12 protruding from the pressing surface 11a against the inner bottom surface 3a of the recess 3. 11a is supported so as to be arranged parallel to the inner bottom surface 3a at a position separated upward from the inner bottom surface 3a of the recess 3. The lid plate 15a of the lid 15 is placed on the sealing material 4a in contact with the sealing material 4a of the culture container 2 by abutting the tip of the protrusion 12 of each cell retainer 10A against the inner bottom surface 3a of the recess 3. Will be done.
In the cell layer preparation tool 1C illustrated in FIG. 7, the protrusion 12 can realize the positioning of each cell holding member 11 in the height direction (vertical direction) with respect to the inner bottom surface regardless of the formation accuracy of the culture container 2 and the lid plate 15a. ..

In the cell layer preparation tool 1C and the lid 15 illustrated in FIG. 7, the cell deposition layer 5 and the culture solution are placed in the recess 3 as shown in FIG. 3, and then the cell holding member 11 is inserted into the recess 3 to form the lid plate. 15a can be placed on the sealing material 4a of the culture vessel 2 and used for preparing a cell layer. The lid 15 on which the lid plate 15a is placed on the sealing material 4a of the culture vessel 2 is removed by moving it upward with respect to the culture vessel 2 after performing cell culture for a culture time of a preset time length. .. If the lid 15 is removed from the culture vessel 2, the plurality of cell holding members 11 inserted in the recess 3 can be taken out from the recess 3 at once.

FIG. 8 illustrates a modified example of the cell layer preparation tool 1D and the cell retainer 10D.
The cell layer preparation tool 1D of FIG. 8 is obtained by changing the cell presser 10A of the cell layer preparation tool 1A of FIGS. 3 to 5 to the cell presser 10D of FIG.
The cell pressing tool 10D of FIG. 8 has a cell pressing member 21 and a pin 22 with a male screw shaft attached to the cell pressing member 21 so that the protrusion dimension from the pressing surface 21a can be changed.

The cell holding member 21 of the cell holding tool 10D of FIG. 8 is formed by forming a female screw hole 21c and a pin accommodating hole 21e in the cell holding member 11 of the cell holding tool 10A described with reference to FIGS. 1 to 5. is there.
The pressing surface 21a of the cell pressing member 21 of the cell pressing member 10D of FIG. 8 has the same configuration as the pressing surface 11a of the cell pressing member 11 described with reference to FIGS. 1 and 3 to 5.

The female screw hole 21c of the cell pressing member 21 of the cell pressing member 10D of FIG. 8 is formed by being recessed from the upper surface 21b opposite to the pressing surface 21a of the cell pressing member 21 toward the pressing surface 21a.
The pin accommodating hole 21e of the cell holding member 21 is formed so as to extend from the bottom of the hole, which is the end of the female screw hole 21c on the holding surface 21a side, toward the holding surface 21a, and the tip side opposite to the base end on the female screw hole 21c side is formed. It is open to the pressing surface 21a. The pin accommodating hole 21e is formed so as to extend coaxially with the female screw hole 21c. Further, the pin accommodating hole 21e is formed to have a smaller diameter than the female screw hole 21c.
The female screw hole 21c and the pin accommodating hole 21e are formed so as to extend perpendicularly to the pressing surface 21a of the cell pressing member 21.

The pin 22 with a male screw shaft has a pin 22a housed in a pin storage hole 21e of the cell holding member 21 and a male screw shaft 22b formed at one end of the pin 22a in the extending direction.
The male screw shaft 22b is formed thicker than the pin 22a.
The male screw shaft 22b is housed in the female screw hole 21c of the cell holding member 21. Further, the male screw shaft 22b is screwed to the cell pressing member 21 by screwing the male screw portion 22c formed on the side surface thereof into the female screw portion 21d on the inner peripheral surface of the female screw hole 21c of the cell pressing member 21.
The pin 22a of the pin 22 with a male screw shaft is formed coaxially with the male screw shaft 22b.

A tool engaging portion 22d for engaging a tool such as a flat-blade screwdriver to rotate the pin 22 with a male screw shaft around the axis is formed at the rear end of the male screw shaft 22b opposite to the tip on the pin 22a side. .. The tool engaging portion 22d illustrated in FIG. 8 is a tool engaging groove capable of engaging the tip of a flat-blade screwdriver.
The male screw shaft 22b illustrated in FIG. 8 is formed in a size that can be accommodated in the female screw hole 21c as a whole. The tool engaging portion 22d is formed at the rear end of the male screw shaft 22b so that the tool can be engaged even when the entire male screw shaft 22b is housed in the female screw hole 21c. The tool engaging portion 22d is recessed from the rear end surface of the male screw shaft 22b, such as a tool engaging groove in which the tip of a flat-blade screwdriver or a Phillips screwdriver can be engaged, for example, a wrench fitting recess in which a hexagon wrench can be inserted and engaged. A tool engagement recess can be adopted. It should be noted that the concave portion may be used as long as it can be engaged with a tool (spanner or the like).

The length of the pin 22a of the pin 22 with a male screw shaft is longer than the total length of the pin accommodating hole 21e of the cell holding member 21.
The portion of the pin 22a that protrudes from the pressing surface 21a of the cell pressing member 21 forms the protrusion 12 of the cell pressing tool 10D.
The cell retainer 10D of FIG. 8 is rotated around the axis by a tool engaged with the tool engaging portion 22d at the rear end of the male screw shaft 22b of the pin 22 with a male screw shaft to position the male screw shaft 22b in the female screw hole 21c axial direction. By changing the position, the position of the pin 22 with a male screw shaft in the direction perpendicular to the pressing surface 21a with respect to the cell pressing member 21 can be adjusted. As a result, the cell pressing tool 10D of FIG. 8 can adjust the protruding dimension of the pin 22a from the pressing surface 21a of the cell pressing member 21, that is, the protruding dimension of the protrusion 12 from the pressing surface 21a.

The male screw shaft 22b of the pin 22 with a male screw shaft and the female screw hole 21c of the cell pressing member 21 form a pin support mechanism that variably supports the pin 22a so as to project from the pressing surface 21a of the cell pressing member 21.
The cell retainer 10D of FIG. 8 can be used for producing the cell layer 6 in place of the cell retainer 10A in the cell layer preparation tools of FIGS. 3 to 5. In the cell pressing tool 10D of FIG. 8, the protruding dimension of the pin 22a from the pressing surface 21a is adjusted in advance, and the cell pressing member 21 is inserted into the recess 3. The cell pressing member 21 inserted into the recess 3 simply abuts the tip of the pin 22a against the inner bottom surface 3a of the recess 3, and the cell pressing member 21 is projected from the inner bottom surface 3a of the recess 3 to the pressing surface 21a of the pin 22a. It can be positioned at a position separated upward by a considerable amount.

The cell pressing tool 10D of FIG. 8 is formed by a cell pressing member 21 having a plurality of pin protrusion amount adjusting holes formed by a female screw hole 21c and a pin accommodating hole 21e, and a plurality of pin protrusion amount adjusting holes of the cell pressing member 21. A configuration having a pin 22 with a male screw shaft inserted in each can be adopted. In the case of this configuration, the pin 22a and the pin support mechanism of the plurality of pins 22 with male screw shafts of the cell presser 10D of FIG. 8 are such that the pressing surface 21a of the cell pressing member 21 is from the inner bottom surface 3a of the recess 3 to the pin 22a. A support mechanism for supporting the cell pressing member 21 can be configured so as to extend parallel to the inner bottom surface 3a at a position separated upward by an amount corresponding to the protrusion dimension from the pressing surface 21a.

The male screw shaft 22b can also adopt a configuration in which an axial dimension that can always protrude from the female screw hole 21c illustrated in FIG. 8 to the upper surface 21b side of the cell holding member 21 is secured.
In this case, it is preferable that the tool engaging portion of the male screw shaft 22b is provided at the rear end portion of the male screw shaft 22b that always protrudes toward the upper surface 21b of the cell holding member 21. Further, the tool engaging portion provided at the rear end portion of the male screw shaft 22b that always projects toward the upper surface 21b side of the cell pressing member 21 is not limited to the tool engaging recess, and for example, a bag wrench, a spanner, or the like is placed on the outside. It may be a rear end engaging portion formed so as to be engaged.

9 and 10 exemplify the cell layer preparation tools 1E and 1F and the cell pressers 10E and 10F of the modified examples. The cell pressers 10E and 10F illustrated in FIGS. 9 and 10 have a presser member 31 with a shaft and an upper support member to which the male screw shaft 33 of the presser member 31 with a shaft is screwed.
The upper support members of the cell retainers 10E and 10F of FIG. 9 are designated by reference numerals 32A, and the upper support members of the cell retainer 10F of FIG. 10 are designated by reference numerals 32B.

The cell layer preparation tool 1E illustrated in FIG. 9 has a culture vessel 2 and a cell retainer 10E including an upper support member 32A placed on the upper surface 4 of the culture vessel 2 and arranged on the recess 3. Is.

The cell presser 10E illustrated in FIG. 9 has a presser member 31 with a shaft and an upper support member 32A to which the male screw shaft 33 of the presser member 31 with a shaft is screwed. The shafted pressing member 31 has a cell pressing member 11 and a male screw shaft 33 protruding from an upper surface 11c on the opposite side of the holding surface 11a of the cell pressing member 11.

The male screw shaft 33 extends from the upper surface 11c of the cell pressing member 11 in a direction perpendicular to the pressing surface 11a to the side opposite to the pressing surface 11a. Further, the male screw shaft 33 is formed so as to extend coaxially with the central axis perpendicular to the holding surface 11a of the cell holding member 11.
It has been postponed. In FIG. 9, the male screw shaft 33 is inserted into the female screw hole 32c of the upper support member 32A. In FIG. 9, the male screw shaft 33 is screwed to the upper support member 32 by screwing the male screw portion 33a formed on the side surface thereof into the female screw portion 32d on the inner peripheral surface of the female screw hole 32c of the upper support member 32A.

The upper support member 32A includes a support member main body 32a extending over the recess 3 and a mounting piece portion 32b placed on a region around the opening of the recess 3 on the upper surface 4 of the culture vessel 2. Have.
As illustrated in FIGS. 11A to 11C, the support member main body 32a can adopt a configuration in which the arm portion 32g extends in a plurality of directions from the central portion 32f through which the female screw hole 32c is formed. The mounting piece portion 32b is formed at the tip of each of the plurality of arm portions 32g of the support member main body 32a. The support member main body 32a is extendedly arranged on the recess 3 by placing the mounting piece portion 32b on the region around the opening of the recess 3 on the upper surface 4 of the culture container 2.
When each mounting piece portion 32b of the upper support member 32A is placed on the upper surface 4 of the culture vessel 2, the female screw hole 32c of the upper support member 32A is oriented so that its central axis extends in the vertical direction.
The space between the arms 32g can be used for inserting the pipette into the clearance 18 between the side surface 11b of the cell holding member 11 and the inner peripheral surface of the recess 3.

The cell retainer 10E is centered on the support member main body 32a by rotating the male screw shaft 33 of the shafted retainer 31 with respect to the upper support member 32A to adjust the screwing position of the male screw shaft 33 with respect to the upper support member 32A. The distance between the portion 32f and the cell holding member 11 can be adjusted.
The cell pressing member 10E can insert the cell pressing member 11 into the recess 3 by adjusting the distance between the central portion 32f of the supporting member main body 32a and the cell pressing member 11 in advance. The upper support member 32A is arranged so as to straddle the opening of the recess 3 by placing the mounting piece portion 32b on the region around the opening of the recess 3 on the upper surface 4 of the culture container 2.
The upper support member 32A placed on the upper surface 4 of the culture container 2 has a cell holding member 21 inserted into the recess 3 by adjusting the distance between the support member main body 32a and the central portion 32f in advance, and the holding surface 21a thereof. Can be supported so as to be arranged parallel to the inner bottom surface 3a at a position separated upward from the inner bottom surface 3a of the recess 3.
The cell retainer 10E can be used for producing a cell layer by supporting the cell retainer 21 inserted in the recess 3 with the upper support member 32A placed on the culture vessel 2 via the male screw shaft 33. ..

The upper support member 32A and the male screw shaft 33 of the shafted pressing member 31 can raise and lower the cell pressing member 21 inserted in the recess 3 with respect to the supporting member main body 32a of the upper supporting member 32A placed on the culture vessel 2. A holding member elevating mechanism that supports the vehicle is configured.
Further, the upper support member 32A and the male screw shaft 33 of the shaft-mounted pressing member 31 have an inner bottom surface of the cell pressing member 21 inserted into the recess 3 at a position where the pressing surface 21a is separated upward from the inner bottom surface 3a of the recess 3. A support mechanism for supporting the cells so as to be arranged parallel to 3a is configured.

In the cell retainer 10F illustrated in FIG. 10, in the cell retainer 10E illustrated in FIG. 9, a through hole 32e having no female screw portion on the inner surface penetrates instead of the upper support member 32A in which the female screw hole 32c is formed through. The formed upper support member 32B is adopted. The through hole 32e is formed in the central portion 32f of the support member main body 32a of the upper support member 32B. The upper support member 32B is different from the upper support member 32A illustrated in FIG. 9 only in that the female screw hole 32c is changed to the through hole 32e.

Further, the cell pressing tool 10F illustrated in FIG. 10 is screwed into a portion of the male screw shaft 33 of the cell pressing member 11 inserted into the through hole 32e of the supporting member main body 32a and protruding upward from the central portion 32f of the supporting member main body 32a. It also has a worn upper nut 34 and a lower nut 35 screwed to a portion protruding downward from the central portion 32f of the support member main body 32a of the male screw shaft 33.
The male screw shaft 33 of the shafted holding member 31 inserted into the through hole 32e of the support member main body 32a rotates the lower nut 35 to change the screwing to the male screw shaft 33, and the upper nut 34 is centered on the support member main body 32a. By tightening to the portion 32f, the separation distance between the central portion 32f of the support member main body 32a and the cell pressing member 11 can be adjusted. Further, the male screw shaft 33 of the holding member 31 with a shaft is fixed to the upper support member 32B by tightening the upper nut 34 to the central portion 32f of the support member main body 32a.

In the cell presser 10F illustrated in FIG. 10, the separation distance between the central portion 32f of the support member main body 32a and the cell presser member 11 is adjusted in advance, and the male screw shaft 33 is fixed to the upper support member 32B to hold the cells. The member 11 can be inserted into the recess 3. Then, in the cell presser 10F, the upper support member 32B is placed on the upper surface 4 of the culture vessel 2, so that the cell presser member 21 inserted into the recess 3 by the upper support member 32B has its pressing surface 21a recessed 3. It can be supported so as to be arranged at a position separated upward from the inner bottom surface 3a of the.

When each mounting piece portion 32b of the upper support member 32B is placed on the upper surface 4 of the culture vessel 2, the through hole 32e of the upper support member 32B has its central axis extending in the vertical direction. The upper support member 32B placed on the upper surface 4 of the culture container 2 can support the cell pressing member 21 inserted in the recess 3 in a direction in which the pressing surface 21a is parallel to the inner bottom surface 3a of the recess 3.
The cell retainer 10F can be used for producing a cell layer by supporting the cell retainer 21 inserted in the recess 3 with the upper support member 32B placed on the culture vessel 2 via the male screw shaft 33. ..

The upper support member 32B and the male screw shaft 33 of the shafted pressing member 31 can raise and lower the cell pressing member 21 inserted in the recess 3 with respect to the supporting member main body 32a of the upper supporting member 32B placed on the culture vessel 2. A holding member elevating mechanism that supports the vehicle is configured.
Further, the upper support member 32B and the male screw shaft 33 of the shaft-mounted pressing member 31 have an inner bottom surface of the cell pressing member 21 inserted into the recess 3 at a position where the pressing surface 21a is separated upward from the inner bottom surface 3a of the recess 3. A support mechanism for supporting the cells so as to be arranged parallel to 3a is configured.

The lid 15 illustrated in FIG. 7 can adopt a configuration in which the cell retainer 10D illustrated in FIG. 8 is used instead of the cell retainer 10A.
Further, as the lid, a lid plate in which female screw holes 32c are formed through at a plurality of locations is adopted, and a configuration in which the cell retainer 10E illustrated in FIG. 9 is assembled at a plurality of locations of the lid plate can also be adopted. As the lid, a lid plate in which through holes 32e are formed through at a plurality of locations is adopted, and a configuration in which the cell retainer 10F illustrated in FIG. 10 is assembled at a plurality of locations of the lid plate can also be adopted.

The cell layer can also be produced by the following method. As illustrated in FIG. 12, cells are placed in the recess 3 on the inner bottom surface 3a of the recess 3 to form a cell deposition layer 5, and the culture solution is placed in the recess 3.

As illustrated in FIG. 13, the cell pressing member 11 of the cell pressing tool 10A is inserted into the recess 3, supported by the protrusion 12 at a position separated upward from the inner bottom surface 3a of the recess 3, and the cell pressing member 11 is pressed. A gap is secured between the surface 11a and the surface 11a.

As illustrated in FIG. 14, the cell deposition layer 5 is cultured while the cell holding member 11 is maintained at a distance from the inner bottom surface 3a of the recess 3 by the protrusion 12, and the cells forming the cell deposition layer 5 are adhered to each other. In addition, the thickness of the cell deposition layer 5 is increased so that the cell deposition layer 5 is brought into surface contact with the pressing surface 11a of the cell pressing member 11. As a result, the cell layer 6 having a constant thickness and being flat is produced in the recess 3. Since the pressing surface 11a is parallel to the inner bottom surface 3a of the recess 3, the upper surface and the bottom surface of the cell layer 6 have parallel surfaces.

The cultured cells may flow out into the gap (clearance 18) between the cell holding member 11 and the entire circumference of the inner peripheral surface 3b of the recess 3. The outflowed cells may be removed by suction with a pipette or the like.

As illustrated in FIG. 15, the cell retainer 10A is taken out of the recess 3 and moved above the recess 3. The prepared cell layer 6 remains on the inner bottom surface 3a of the recess 3. Since the upper surface of the cell layer 6 is flat (a plane suitable for observation), it is suitable for observation with a microscope or the like.

Examples of the cell non-adhesive material of the cell holding member include, but are not limited to, glass and PDMS (Poly Dimethicylpolysiloxane), and a cell non-adhesive material known to those skilled in the art can be used. .. The cell pressing member may be one in which the pressing surface in contact with the cells is coated with the above-mentioned cell-adhesive material.

The cell deposition layer 5 and the culture solution are housed in the recess 3 of the culture container 2. The cells housed in the recess 3 may be any cells as long as they can adhere to each other to form the cell layer 6, and for example, animal-derived cells and plant-derived cells. And so on.

Examples of animal species from which cells are derived include mammals, birds, reptiles, amphibians, fish, and insects. Mammals include, for example, humans, chimpanzees, marmosets, dogs, cats, mice, rats, rabbits, monkeys, pigs, goats, sheep, cows, ferrets and the like.

The cells may be pluripotent stem cells, established cells such as cancer cells, and the like. Examples of the established cells include, but are not limited to, human normal heart fibroblasts (NHCF), human normal skin fibroblasts (NHDF), human umbilical vein endothelial cells (HUVEC), NIH3T3 cells, and the like. .. Examples of pluripotent stem cells include tissue stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like.

The cell may be a cell isolated from tissue. The tissue may be adipose tissue, skin tissue, liver tissue, renal tissue, vascular tissue or the like.

The cell layer 6 is composed of a single layer or a plurality of layers, and the thickness of the cell layer 6 is equal to or greater than the thickness of one cell. The thickness of the cell layer 6 is 3 μm or more, 5 μm or more, 10 μm or more, 15 μm or more, 20 μm or more, 25 μm or more, 30 μm or more, 35 μm or more, 40 μm or more, 45 μm or more, 50 μm or more, 55 μm or more, 60 μm or more, 65 μm or more, The thickness may be 70 μm or more, 75 μm or more, 80 μm or more, 85 μm or more, 90 μm or more, 95 μm or more, 100 μm or more, or more.

The force for pressing the cell deposition layer 5 when producing the cell layer 6 can be set to an appropriate value in advance depending on the type of cells and the like. The force for pressing the cell deposition layer 5 is preferably set within a range that does not adversely affect the survival of cells and the physiological function of cells.

The cells may be cultured in a container containing the cells and the culture solution in an environment _{of, for example, 37 ° C., 100% humidity, and 5% CO 2 concentration.} Conditions such as temperature, humidity, and CO ₂ concentration can be changed as appropriate.

Examples of the basal medium used for culturing cells include, but are not limited to, Eagle's medium, Dalveco's minimum medium, F10 medium, F12 medium, RPMI1640 medium, L-15 medium and the like.

The medium used for culturing the cells may be the above-mentioned basal medium supplemented with serum. Examples of the serum added to the culture solution include bovine serum, fetal bovine serum, horse serum, human serum and the like.

The present invention includes the following aspects.
[1] A cell pressing member having a non-cell adhesive region and inserted into a recess of a culture vessel and the cell pressing member inserted into the recess have a pressing surface at the lower end thereof upward from the inner bottom surface of the recess. A cell retainer having a support mechanism that supports the recess so as to be arranged parallel to the inner bottom surface of the recess at a position separated from the cell.
[2] The cell presser according to [1], wherein the support mechanism is a protrusion projecting from the cell presser member and abutting against the culture vessel from above.
[3] The cell retainer according to [2], wherein the protrusion is projected from the pressing surface of the cell retainer in a pin shape and the tip is abutted against the inner bottom surface of the recess.
[4] The protrusion is a portion of a pin supported by the cell pressing member that protrudes from the pressing surface, and the supporting mechanism is provided on the pin and the cell pressing member, and the pin is attached to the cell pressing member. The cell presser according to [3], which has a pin support mechanism that variably supports the protrusion dimension from the presser surface.
[5] The support mechanism is a spacer projection that protrudes from the side surface of the cell pressing member and comes into contact with the inner peripheral surface of the culture vessel, and the spacer projection is the circumferential direction of the side surface of the cell pressing member. The cell presser according to any one of [1] to [3], which secures a clearance between the cell presser member inserted into the recess and the entire inner peripheral surface of the recess. Ingredients.
[6] The support mechanism is placed on a region around the opening of the recess in the culture vessel and the support member main body extending over the recess so that the support member main body is placed on the recess. It has an upper support member having a mounting piece portion that supports the cells, and a pressing member supporting mechanism that supports the cell pressing member so as to be able to move up and down with respect to the supporting member main body of the upper supporting member arranged on the recess. , [1] or [5].
[7] A cell layer preparation tool comprising a culture container and a lid for opening and closing the opening of a recess of the culture container, wherein the lid is provided with the cell retainer according to any one of [1] to [4].
[8] The cell layer preparation tool according to [7], wherein a plurality of the recesses are formed in the culture container, and the lid includes the plurality of cell holders.
[9] The cell layer preparation tool according to [7] or [8], wherein cells are housed inside the recess.
[10] The cells placed in the recess are deposited on the inner bottom surface of the recess of the culture vessel to form a cell deposition layer, and the cell retainer of the cell retainer according to any one of [1] to [4]. The member is inserted into the recess, supported by the support mechanism at a position upwardly separated from the inner bottom surface of the recess, the cell deposition layer is pressed by the cell pressing member, and the cell deposition layer is pressed by the cell pressing member. The cells that are brought into surface contact with the pressing surface of the member and that the cells are cultured by the supporting mechanism while maintaining the distance from the inner bottom surface of the recess to form the cell deposition layer. A method for producing a cell layer, including adhering to each other.
[11] The cells placed in the recess are deposited on the inner bottom surface of the recess of the culture vessel to form a cell deposition layer, and the cell retainer of the cell retainer according to any one of [1] to [4]. The member is inserted into the recess and supported by the support mechanism at a position upwardly separated from the inner bottom surface of the recess to secure a gap between the pressing surface of the cell pressing member and the cell deposition layer. In addition, the cells are cultured while the cell holding member is maintained at a distance from the inner bottom surface of the recess by the support mechanism, and the cells forming the cell deposition layer are adhered to each other, and the cell deposition is performed. A method for producing a cell layer, which comprises increasing the thickness of the layer to bring the cell deposition layer into surface contact with the pressing surface of the cell pressing member.

1A ... Cell layer preparation tool, 1B ... Cell layer preparation tool, 1C ... Cell layer preparation tool, 1D ... Cell layer preparation tool, 1E ... Cell layer preparation tool, 1F ... Cell layer preparation tool, 2 ... Culture container, 3 ... Recess 3, 3a ... Inner bottom surface, 3b ... Inner peripheral surface, 4 ... Top surface, 4a ... Sealing material, 5 ... Cell deposition layer, 6 ... Cell layer, 10A ... Cell presser, 10B ... Cell presser, 10D ... Cell presser, 10E ... Cell presser, 10F ... Cell presser, 11 ... Cell presser, 11a ... Pressing surface, 11b ... Side surface, 11c ... Top surface, 12 ... Projection, 13 ... Spacer projection, 13a ... Vertical bar, 13b ... Horizontal bar , 14 ... side protrusion, 15 ... lid, 15a ... lid plate, 18 ... clearance, 21 ... cell holding member, 21a ... holding surface, 21b ... upper surface, 21c ... female screw hole, 21d ... female screw part, 21e ... pin accommodating hole , 22 ... Pin with male screw shaft, 22a ... Pin, 22b ... Male screw shaft, 22c ... Male screw part, 22d ... Tool engaging part, 31 ... Holding member with shaft, 32A ... Upper support member, 32B ... Upper support member, 32a ... Support member body, 32b ... mounting piece, 32c ... female screw hole, 32d ... female screw, 32e ... through hole, 32f ... central, 32g ... arm, 33 ... male screw shaft, 33a ... male screw, 34 ... upper nut , 35 ... Lower nut.

International Publication No. 2017/077985

Claims (11)

A cell holding member that has a non-adhesive region and is inserted into the recess of the culture vessel,
A support mechanism that supports the cell pressing member inserted into the recess so that the pressing surface at the lower end thereof is arranged parallel to the inner bottom surface of the recess at a position separated upward from the inner bottom surface of the recess.
Cell presser with. The cell presser according to claim 1, wherein the support mechanism is a protrusion projecting from the cell presser member and abutting against the culture vessel from above. The cell presser according to claim 2, wherein the protrusion is projected from the pressing surface of the cell presser in a pin shape, and the tip is abutted against the inner bottom surface of the recess. The protrusion is a portion of a pin supported by the cell holding member that protrudes from the holding surface.
The cell presser according to claim 3, wherein the support mechanism includes the pin and a pin support mechanism provided on the cell presser member to variably support the pin so as to project from the presser surface of the cell presser member. Ingredients. The support mechanism is a spacer projection that protrudes from the side surface of the cell pressing member and abuts on the inner peripheral surface of the culture vessel, and the spacer projection is a plurality of locations in the circumferential direction of the side surface of the cell pressing member. The cell presser according to any one of claims 1 to 3, wherein a clearance is secured between the cell presser member dispersedly arranged in the recess and inserted into the recess and the entire inner peripheral surface of the recess. The support mechanism supports the support member main body on the recess by being placed on a region around the opening of the recess in the culture vessel and the support member main body extending over the recess. A claim that includes an upper support member having a mounting piece portion, and a pressing member supporting mechanism that supports the cell pressing member so as to be able to move up and down with respect to the supporting member main body of the upper supporting member arranged on the recess. The cell retainer according to 1 or 5. A cell layer preparation tool comprising a culture container and a lid for opening and closing the opening of a recess of the culture container, wherein the lid includes the cell retainer according to any one of claims 1 to 4. The cell layer preparation tool according to claim 7, wherein a plurality of the recesses are formed in the culture container, and the lid includes the cell holders. The cell layer preparation tool according to claim 7 or 8, wherein the cells are housed in the recess. To form a cell deposition layer by depositing cells placed in the recess on the inner bottom surface of the recess of the culture vessel.
The cell holding member of the cell holding tool according to any one of claims 1 to 4 is inserted into the recess and supported by the supporting mechanism at a position upwardly separated from the inner bottom surface of the recess. The cell deposition layer is pressed by the cell pressing member, and the cell deposition layer is brought into surface contact with the pressing surface of the cell pressing member.
By culturing the cells while maintaining the distance between the recesses and the inner bottom surface of the cell holding member by the support mechanism, the cells forming the cell deposition layer are adhered to each other.
A method for producing a cell layer, including. To form a cell deposition layer by depositing cells placed in the recess on the inner bottom surface of the recess of the culture vessel.
The cell holding member of the cell holding tool according to any one of claims 1 to 4 is inserted into the recess and supported by the supporting mechanism at a position upwardly separated from the inner bottom surface of the recess. To secure a gap between the holding surface of the cell holding member and the cell deposition layer, and
The cells are cultured while the cell holding member is maintained at a distance from the inner bottom surface of the recess by the support mechanism, and the cells forming the cell deposition layer are adhered to each other, and the thickness of the cell deposition layer is formed. To bring the cell deposition layer into surface contact with the holding surface of the cell holding member,
A method for producing a cell layer, including.

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