JP6081276B2 - Cell culture carrier - Google Patents

Description

  The present invention relates to a cell culture carrier that can be suitably used for culturing mesenchymal stem cells as cell aggregates.

  Mesenchymal stem cells are undifferentiated cells present in bone marrow, adipose tissue, etc., and are known to have the ability to differentiate into mesodermal cells such as bone cells, adipocytes, chondrocytes, etc. It has been. For this reason, mesenchymal stem cells are expected to be used for regenerative medicine, and clinical studies on cell therapy for transplanting mesenchymal stem cells to sites where self-renewal is difficult are being conducted.

By the way, although cartilage cells can be obtained by inducing differentiation from mesenchymal stem cells as described above, it is necessary to form cell aggregates. However, the mesenchymal stem cells have a problem that they do not have a property of self-aggregating by adhering and growing in a flat shape on a petri dish.
Therefore, in Patent Document 1, in order to obtain a large amount of mesenchymal stem cell aggregates having a uniform differentiation state, a culture carrier having a three-dimensional shape having a plurality of recesses (hereinafter referred to as wells). A method for inducing differentiation into a cartilage tissue after culturing with the use of is used.

FIG. 23 schematically shows the cell culture carrier described in Patent Document 1.
FIG. 23 (a) is a plan view of the cell culture carrier, and FIG. 23 (b) is a sectional view taken along the line CC in FIG. 23 (a). FIG. 23C is a cross-sectional view showing a state in which the cell culture carrier is accommodated in the culture container.
The illustrated cell culture carrier 50 is formed of a plate-like zirconia ceramic sintered body, and its upper surface 51 has a predetermined roughness (root mean square roughness Rq of 100 nm to 280 nm) and a linear density per 1 μm length. The number of wells 52 is 1.6 to 3.0.
Further, the opening of each well 52 is circular or rectangular (only the circular shape is shown in the figure), the opening diameter is 70 μm to 550 μm, and the distance between the central points of the adjacent wells is 80 μm to 700 μm. Is formed.

In order to culture cartilage cells using the cell culture carrier 50, first, the cell culture carrier 50 is accommodated in a plastic container 60 as shown in FIG. Subsequently, as shown in FIG. 24A, the plastic container 60 is accommodated in a plastic case 61, and a first culture solution (medium) for culturing mesenchymal stem cells in the plastic container 60 is used. 62 is supplied and allowed to stand for a predetermined time.
The culture solution (medium) placed in the plastic container 60 enters the cell culture carrier 50 by capillary action, and the well 52 is filled with the first culture solution (medium) 62.

Thereafter, as shown in FIG. 24B, the inside of the plastic container 60 is filled with a second culture solution (medium) 63 containing mesenchymal stem cells C and allowed to stand for a predetermined time. By standing still, the mesenchymal stem cells in the plastic container 60 settle by gravity and deposit in the well 52 and the upper surface 51. A microscope photograph in this state is shown in FIG.
And as shown in FIG.24 (c), the 1st culture solution (medium) 62 is added in the said plastic container 60, the upper surface of the cell culture support | carrier 50 is covered, and it leaves still for a predetermined time.

As a result, the mesenchymal stem cell C deposited on the upper surface 51 of the cell culture carrier 50 falls into the well 52, and the aggregation of the mesenchymal stem cell proceeds in the well 52. Aggregates are formed. A microscope photograph showing this state is shown in FIG.
After the culture solution (medium) in the plastic container 60 and the cell culture carrier 50 is sucked with an aspirator, the mesenchymal stem cells are turned into chondrocytes in the cell culture carrier 50 as shown in FIG. A third culture solution (medium) 64 containing a low molecular weight compound or protein that induces differentiation is added and left to stand for a predetermined time. FIG. 27 shows a microscope photograph showing the initial state after standing, and FIG. 28 shows a microscope photograph showing the intermediate state.
Thereafter, the aggregated mesenchymal stem cells differentiate into chondrocytes.

JP 2012-50426 A

By the way, since the cell culture carrier 50 is disposed and used in a plastic container (culture container) 60, cells escape (enter) into the gap between the plastic container (culture container) 60 and the cell culture carrier 50, and between each well 52. There was a problem that the cell concentration of the cells became non-uniform.
In addition, as shown in FIG. 24B, when the second culture solution (medium) 63 containing mesenchymal stem cells is placed in the plastic container (culture container) 60 and allowed to stand for a predetermined time, the cell culture carrier All of the mesenchymal stem cells C deposited on the upper surface 51 of 50 do not fall into the well 52, but near the boundary (corner portion) between the upper surface 51 and the side wall 61 of the plastic container (culture container) 60. The cells C seeded in (1) are deposited (see FIG. 25).
Thereafter, as shown in FIG. 24 (c), when the first culture solution (medium) is supplied and allowed to stand for a predetermined time, cells gather in a ring shape, and a ring-shaped cell aggregate R is formed. (See FIG. 26). In FIG. 26, a part of the ring-shaped cell aggregate R is peeled off.

In addition, as shown in FIG. 24 (d), the third culture containing a low molecular compound or protein that induces differentiation of mesenchymal stem cells into chondrocytes in the cell culture carrier 50 in the plastic container (culture container) 60. When the mesenchymal stem cells C are differentiated into chondrocytes by adding the liquid (medium) 64, the ring-shaped cell aggregates are detached from the vicinity (corner) of the boundary, and the mesenchymal stem cells C in the well 52 are taken up. Then, the ring diameter is narrowed and condensed on the upper surface 51 of the cell culture carrier 50 in the form of flakes to differentiate into chondrocytes 65 (see FIGS. 27 and 28).
Finally, as shown in FIG. 24 (e), the mesenchymal stem cells C (chondral cells after the change) in the well 52 almost disappear, and one large flaky chondrocyte in the center of the upper surface 51. 65 is formed (see FIG. 29).

Thus, even when the cell culture carrier disclosed in Patent Document 1 is used, the above-described phenomenon occurs at a relatively high frequency, and the cell coagulation of mesenchymal stem cells having a uniform differentiation state with a high probability. It turned out to be difficult to obtain a large number of aggregates. In addition, since the culture vessel and the cell culture carrier are formed separately as in the prior art, there is a problem that a gap is formed between the two and the cell concentration is non-uniform.
Therefore, in order to solve this technical problem, the present inventors have further improved the invention described in Patent Document 1 to suppress ring-shaped cell aggregates and to increase the size with higher probability. The present inventors have intensively studied to form uniform cell aggregates and arrived at the present invention.

  An object of the present invention is to provide a cell culture carrier capable of aggregating mesenchymal stem cells in a uniform shape and obtaining a cell aggregate with a uniform differentiation state with high probability.

  The cell culture support according to the present invention made to solve the above problems is formed in a container shape having a bottom wall portion and a side wall portion provided on a peripheral edge of the bottom wall portion, and is formed on the bottom wall portion. A cell culture carrier used for culturing mesenchymal stem cells, in which a plurality of wells are formed, comprising a ceramic porous body having an average pore diameter of 0.1 μm to 0.6 μm and a porosity of 40% to 55% And the inner peripheral surface of the side wall portion is formed so as to expand toward the upper side with an inclination angle of 100 ° to 135 ° with respect to the upper surface of the bottom wall portion, and 2 on the upper surface of the bottom wall portion. The mean square roughness Rq is 100 nm or more and 280 nm or less, and the linear density per 1 μm length is 1.6 or more and 3.0 or less, and the openings of the plurality of wells have a circular shape having a uniform diameter. The bottom of the well The well has a diameter of 100 μm or more and 550 μm or less, and a cross-sectional shape of the bottom of the well is formed in a curved shape having a concave curve, and at least one of the plurality of wells is a boundary between the bottom wall and the side wall. One well that is formed at a position in contact with the line, or straddles the boundary line, and the horizontal dimension of the side wall part is at least twice as long as the penetration dimension of the well into the side wall part. The distance dimension on the central line connecting the center of the well from the outer peripheral line of the other opening to the outer peripheral line of the opening of another well is 10 μm or more and 50 μm or less.

  In the cell culture carrier thus configured, mesenchymal stem cells can be aggregated in a uniform shape, and cell aggregates with a uniform differentiation state can be obtained with high probability.

  In particular, since the cell culture carrier is formed in a container shape, there is no need for a plastic container that accommodates the carrier at the time of culture, and there is no gap generated when the plastic container and the carrier are formed separately as in the prior art. Therefore, the cell concentration between the wells can be made uniform.

The cell culture carrier according to the present invention is composed of a porous ceramic body having an average pore diameter of 0.1 μm to 0.6 μm and a porosity of 40% to 55%.
When the average pore diameter is less than 0.1 μm or the porosity is less than 40%, the cultured cells do not aggregate three-dimensionally and may adhere to the ceramic porous body in a flat shape, which is not preferable.
Further, when the average pore diameter exceeds 0.6 μm or the porosity exceeds 55%, the strength of the ceramic porous body becomes insufficient, and the ceramic particles easily peel off. The peeled particles are put in the wells. Since the cell aggregate to form takes in, it is not preferable.

In addition, since the inner peripheral surface of the side wall portion is formed to expand as it goes upward with an inclination angle of 100 ° to 135 ° with respect to the upper surface of the bottom wall portion, the mesenchymal stem cells in a ring shape The formation of aggregates can be suppressed.
When the inner peripheral surface of the side wall portion is less than 100 ° with respect to the top surface of the bottom wall portion, cells seeded in (corner portion) are deposited near the boundary line between the bottom wall portion and the side wall portion, and ring-shaped Since cell aggregates are formed on the surface, it is not preferable.
On the other hand, when the inner peripheral surface of the side wall part exceeds 135 ° with respect to the upper surface of the bottom wall part, mesenchymal stem cells adhere to the side wall part and deposit near the boundary between the bottom wall part and the side wall part. This is not preferable because cell aggregates are formed in a ring shape.

  Further, since the mean square roughness Rq of the upper surface of the bottom wall portion is 100 nm or more and 280 nm or less and the linear density per 1 μm length is 1.6 or more and 3.0 or less, the flattened mesenchyme The stem cells do not adhere to the upper surface, maintain a spherical shape, and can finally enter the nearest well. In addition, even when mesenchymal stem cells adhere to the upper surface and become flattened in the initial stage of mesenchymal stem cell culture, the mesenchymal stem cells that have become flattened become spherical after a certain amount of time has passed. .

In addition, the openings of the plurality of wells have a circular shape having a uniform diameter, and the bottom of the well has a diameter of 100 μm or more and 550 μm or less, and the cross-sectional shape of the bottom of the well is formed into a curved shape that is concavely curved. Has been.
The openings of the plurality of wells have a circular shape having a uniform diameter, whereby the size of the obtained mesenchymal stem cell aggregate can be made more uniform. For this purpose, the diameter of the bottom of each well is preferably uniform.
The cells formed in the well are determined by the diameter of the bottom of the well. When the diameter of the bottom of the well is 100 μm or more and 550 μm or less, the number of cells is large and the homogeneity of each cell is high. Mesenchymal stem cell aggregates can be obtained.
Furthermore, since the cross-sectional shape of the bottom of the well is formed into a curved shape that is concavely curved, the formation of a plurality of cell aggregates is suppressed in the well, and one cell aggregate is cultured in each well. And the size of the cell aggregates between the wells can be made uniform.

Further, the cell culture carrier is formed such that at least one of the plurality of wells is in contact with a boundary line between the bottom wall part and the side wall part, or straddles the boundary line, and the side wall part Since the horizontal dimension of the cell has a length more than twice the dimension of the well entering the side wall, the cells in the vicinity of the boundary line between the bottom wall and the side wall can be dropped into the well. And the formation of ring-shaped mesenchymal stem cell aggregates can be suppressed.
In addition, even if cells gather near the boundary between the bottom wall and the side wall, the well can divide ring-shaped mesenchymal stem cell aggregates, and ring-shaped mesenchymal stem cell aggregates. Aggregate growth can be suppressed.

Here, when the horizontal dimension of the side wall portion is less than twice the penetration size of the well into the side wall portion, cells in the cell turbid solution are unlikely to collect in the well and the well close to the side wall portion, and the generated cells This is not preferable because the aggregates are smaller than those of other wells and uniform cell aggregates cannot be generated.
This function and effect is more remarkable and more reliable, combined with the configuration in which the inclination angle of the inner peripheral surface of the side wall portion is not less than 100 ° and not more than 135 °.

Further, since the distance dimension on the center line connecting the centers of the wells between the outer peripheral lines of the adjacent well openings (between the inner peripheral surfaces) is 10 μm or more and 50 μm or less, almost all the mesenchymal stem cells are removed from the bottom wall It is possible to enter the inside of the well without adhering to.
That is, when the distance dimension on the center line connecting the centers of the wells between the outer peripheral lines of the adjacent wells is less than 10 μm, the strength between the wells is insufficient, the wells cannot be arranged, and the distance When the size exceeds 50 μm, the mesenchymal stem cells do not completely enter the well portion and adhere to the bottom wall portion, which is not preferable.

Further, the upper surface of the bottom wall portion surrounded by the side wall portion is circular, and at least six wells are formed on a boundary line between the side wall portion and the bottom wall portion, and at least six wells are formed. The center of each of the two wells is arranged at an intersection of a plurality of straight lines passing through the center of the upper surface of the bottom wall portion and a boundary line between the side wall portion and the bottom wall portion, and two wells adjacent to each other on the boundary line. It is desirable that an angle formed by the straight line passing through the center is 60 ° or less.
Thus, by providing at least six wells on the boundary line, formation of ring-shaped cell aggregates generated in the vicinity of the boundary line on the side wall part and the bottom wall part can be more reliably suppressed. .

The plurality of wells formed on the upper surface of the bottom wall portion is preferably arranged in a planar hexagonal lattice shape.
Thus, by arranging a plurality of wells in a hexagonal lattice shape, the distance between all adjacent wells becomes substantially equal, and the uniformity of the size of the formed cells can be improved.

  According to the present invention, mesenchymal stem cells can be aggregated in a uniform shape, and cell aggregates with a uniform differentiation state can be obtained with high probability.

Fig.1 (a) is a top view which shows typically 1st Embodiment of the cell culture carrier which concerns on this invention, FIG.1 (b) is the II arrow sectional drawing. 2A is a cross-sectional view showing a modification of the bottom shape of the well of the cell culture carrier of FIG. 1, and FIG. 2B is a cross-sectional view showing another modification of the bottom shape of the well. is there. FIG. 3 is a plan view for explaining the distance between adjacent wells in a plurality of wells arranged on the upper surface of the bottom wall portion. FIG. 4 is a plan view for explaining a state where the well is in contact with the boundary line between the inner peripheral surface of the side wall and the bottom wall in the cell culture carrier of FIG. 5A and 5B are diagrams for explaining a state in which the well has entered the side wall in the cell culture carrier of FIG. 1, wherein FIG. 5A is a plan view, and FIG. 5B is a cross-sectional view taken along the line II of FIG. is there. FIG. 6 is a cross-sectional view showing an undesirable formation position and an undesirable shape of the well. FIG. 7 is a plan view for explaining preferable conditions when a plurality of wells are arranged on the boundary line between the upper surface of the bottom wall portion and the side wall portion. FIG. 8 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and shows a state in which a cell culture carrier is housed in a case. FIG. 9 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and shows a state in which the first culture solution is supplied into the case. FIG. 10 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and shows a state in which the first culture solution has entered the cell culture carrier. FIG. 11 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and is a view showing a state in which a second culture solution is supplied to a cell culture carrier. FIG. 12 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and showing a state in which mesenchymal stem cells fall on the bottom wall portion of the cell culture carrier. is there. FIG. 13 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and shows a state in which the first culture solution is supplied to the cell culture carrier. FIG. 14 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and showing a state in which cell aggregates are generated in the wells of the cell culture carrier. . FIG. 15 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and shows a state in which the cell culture carrier and the culture medium in the case are removed by an aspirator. . FIG. 16 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and shows a state in which a third culture solution for converting mesenchymal stem cells into chondrocytes is supplied. FIG. FIG. 17 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and shows a state in which the mesenchymal stem cells are changed to chondrocytes. FIG. 18 is a schematic diagram for explaining a process of culturing mesenchymal stem cells and inducing differentiation into chondrocytes, and is a diagram showing a state in which the third culture solution is discharged and chondrocytes are collected. . FIG. 19 is a microscope photograph at the end of the process shown in FIG. 12, focusing on the bottom wall of the cell culture carrier. FIG. 20 is a microscope photograph at the end of the process shown in FIG. 14, focusing on the bottom wall of the cell culture carrier. FIG. 21 is a microscope photograph at the end of the step shown in FIG. 14, focusing on the well of the cell culture carrier. FIG. 22 is a microscope photograph at the end of the process shown in FIG. 18, focusing on the well of the cell culture carrier. FIG. 23 is a diagram schematically showing a conventional cell culture carrier, where (a) is a plan view, (b) is a cross-sectional view taken along the line CC of (a), and (c) is housed in a container. It is sectional drawing which shows a state. FIG. 24 is a schematic diagram for explaining a process of culturing mesenchymal stem cells using a conventional cell culture carrier and inducing differentiation into chondrocytes. FIG. 25 is a microscope photograph showing a state in which the mesenchymal stem cells filled with the second culture medium (medium) containing mesenchymal stem cells are sedimented by gravity and deposited in the well and on the upper surface. FIG. 6 is a diagram focusing on the bottom wall of the cell culture carrier. FIG. 26 is a microscope photograph showing a state in which agglomeration of mesenchymal stem cells has progressed and cell aggregates are formed in the wells, focusing on the bottom wall of the cell culture carrier. . FIG. 27 is a microscope photograph showing an initial state in which a third culture solution (medium) containing a low molecular weight compound or protein that induces differentiation of mesenchymal stem cells into chondrocytes is placed and allowed to stand. It is the figure which focused on the bottom wall part of the support | carrier. FIG. 28 is a microscope photograph showing a medium state in which a third culture solution (medium) containing a low molecular weight compound or protein that induces differentiation of mesenchymal stem cells into chondrocytes is placed and allowed to stand; It is the figure which focused on the bottom wall part of the support | carrier. FIG. 29 is a microscope photograph showing the generated chondrocytes, focusing on the bottom wall of the cell culture carrier.

  Hereinafter, embodiments of a cell culture carrier according to the present invention will be described with reference to the drawings. The cell culture carrier according to the present invention is a cell culture carrier formed in a container shape used for culturing mesenchymal stem cells, and has a plurality of wells (concave portions) for culturing cells on the upper surface of the bottom wall portion. Is formed. 1A is a plan view schematically showing the cell culture carrier according to the present invention, and FIG. 1B is a cross-sectional view taken along the line AA.

A cell culture carrier 1 shown in FIGS. 1 (a) and 1 (b) is made of a ceramic sintered body, and has a disk-like bottom wall portion 2 and a side wall portion 3 erected from the periphery thereof. The upper part is formed in a container shape.
As the ceramic material forming the cell culture carrier 1, zirconia, titania, alumina, hydroxyapatite, or the like can be used as a material that is excellent in biocompatibility and biocompatibility and is suitable as a cell scaffold. . Among these, in the present invention, it is more preferable to use zirconia because cell aggregates of mesenchymal stem cells can be efficiently formed.

The cell culture carrier 1 is composed of a ceramic porous body having an average pore diameter of 0.1 μm to 0.6 μm and a porosity of 40% to 55%.
When the average pore diameter is less than 0.1 μm or the porosity is less than 40%, the cultured cells do not aggregate three-dimensionally and may adhere to the ceramic porous body in a flat shape, which is not preferable. Further, when the average pore diameter exceeds 0.6 μm or the porosity exceeds 55%, the strength of the ceramic porous body becomes insufficient, and the ceramic particles easily peel off. The peeled particles are put in the wells. Since the cell aggregate to form takes in, it is not preferable.
The average pore diameter is measured according to JIS R 1655 (2003), and the porosity is measured according to JIS R 1634 (1998).

The inner peripheral surface 3 a of the side wall portion 3 is inclined so as to expand toward the upper side with respect to the upper surface of the bottom wall portion 2. More specifically, as shown in FIG. 1B, the inclination angle θ1 of the inner peripheral surface 3a is set to an angle of 100 ° to 135 °.
By providing the inclined inner peripheral surface 3a in this way, mesenchymal stem cells in the culture solution do not concentrate near the boundary line 6 between the inner peripheral surface 3a and the upper surface of the bottom wall portion 2, and in the vicinity of the boundary line 6 Generation of ring-shaped cell aggregates can be suppressed.
A horizontal side wall upper surface 3b is formed at the upper end of the inner peripheral surface 3a.

When the inclination angle θ1 of the inner peripheral surface 3a of the side wall part 3 is less than 100 ° with respect to the upper surface of the bottom wall part 2, it is located near the boundary line 6 between the bottom wall part 2 and the side wall part 3 (corner part). Since the seeded cells are deposited, the cells are likely to gather in a ring shape, and the probability that a ring-shaped cell aggregate is generated is not preferable.
On the other hand, when the inclination angle θ1 of the inner peripheral surface 3a of the side wall part 3 exceeds 135 ° with respect to the upper surface of the bottom wall part 2, mesenchymal stem cells adhere to the inner peripheral surface 3a of the side wall part 3, These mesenchymal stem cells are deposited near the borderline 6 (corner).
Thus, since mesenchymal stem cells are collected in a ring shape, the probability that a ring-shaped cell aggregate is generated is increased, which is not preferable.

The top surface of the bottom wall portion 2 is in a surface state having a root mean square roughness Rq of 100 to 280 nm and a linear density per length of 1 μm of 1.6 to 3.0. A plurality of wells 4 are formed in the wall 2.
Since the upper surface of the bottom wall 2 is formed in such a surface state, flattened mesenchymal stem cells do not adhere to the upper surface, maintain a spherical shape, and finally enter the nearest well Can do.
In addition, even when mesenchymal stem cells adhere to the upper surface and become flattened in the initial stage of mesenchymal stem cell culture, the mesenchymal stem cells that have become flattened become spherical after a certain amount of time has passed. .

Moreover, since a plurality of wells 4 are formed on the upper surface of the bottom wall portion 2, spherical mesenchymal stem cells migrate to and aggregate in the wells 4 to efficiently form aggregates of mesenchymal stem cells, It is possible to efficiently induce differentiation of hyaline chondrocyte tissue cells from mesenchymal stem cells.
In addition, when the root mean square roughness Rq and the linear density are outside the above numerical ranges, the cells tend to have a flat shape and adhere to the upper surface of the carrier and tend not to form cell aggregates.

Here, the root mean square roughness Rq and the linear density are parameters for defining the surface roughness. The linear density here is a parameter in the surface direction, and represents the number of times the roughness curve per 1 μm length intersects the average surface.
The mean square roughness Rq in the present invention is measured according to JIS B0601. The linear density is measured with an atomic force microscope (AFM) at a scale of 0.8 μm and a scan size of 10 μm × 10 μm.

Each of the wells 4 has a circular shape in the plan view of the opening, and the diameter of the upper portion of the opening is 100 μm or more and 550 μm or less. The diameter of the upper part of the opening is more preferably 200 μm or more and 550 μm or less.
The shape of the well opening is preferably a circular shape from the viewpoint of easy seeding of cells and formation of cell bodies, and well processability.

Moreover, when the upper part of the opening part of said several well has a uniform diameter, the magnitude | size of the mesenchymal stem cell aggregate obtained can be made more equal.
Furthermore, when this cell culture carrier 1 having a well opening diameter in the above range is cultured to induce differentiation of mesenchymal stem cell aggregates into tissue cells such as chondrocytes, adipocytes, and osteoblasts, this carrier. In one well 4, differentiation can be induced while controlling the size of the mesenchymal stem cell aggregate as it is.
When the opening diameter of the well 4 is less than 100 μm or more than 550 μm, it becomes difficult for the mesenchymal stem cells to form a desired three-dimensional cell aggregate.

Further, the bottom of each well 4 is formed in a hemispherical shape that is concavely curved as shown in FIG. The bottom has a diameter of 100 μm or more and 550 μm or less. More preferably, the bottom portion has a diameter of 200 μm or more and 550 μm or less.
The cross-sectional shape of the bottom of the well 4 is not limited to the hemispherical shape (semicircular shape) as described above, but may be any curved shape that is curved in a concave shape. For example, as shown in FIG. Alternatively, it may be a cross-sectional shape with only the bottom portion loosely and sharply pointed, or a cross-sectional shape that is loosely pointed with a concave shape while reducing the diameter from the opening toward the bottom as shown in FIG.
The depth of the well 4 is preferably 100 μm to 550 μm in the same manner as the opening diameter of the well 4 from the viewpoint of controlling the size of the cell aggregate.

Further, the cells formed in the well 4 are determined by the diameter of the bottom of the well 4, but when the diameter of the bottom of the well 4 is 100 μm or more and 550 μm or less, the number of cells is large and each cell is homogeneous. Highly mesenchymal stem cell aggregates can be obtained. Furthermore, by setting the diameter of the bottom of the well 4 to 200 μm or more, a mesenchymal stem cell aggregate with higher homogeneity of each cell can be obtained.
In addition, as described above, since the cross-sectional shape of the bottom of the well 4 is formed in a curved shape that is concavely curved, the formation of a plurality of cell aggregates in the well is suppressed, and one cell is formed in each well 4. Aggregates can be cultured, and the size of cell aggregates between the wells 4 can be made uniform.

The diameter of the upper portion of the well 4 may be the same as the diameter of the bottom of the well, that is, the well 4 in which the inner wall of the well 4 is vertical, and the diameter of the opening of the well 4 is equal to that of the well bottom. The well 4 may be larger than the diameter (preferably 1.05 times or 1.20 times), and the inner wall (inner peripheral surface) of the well 4 may be tapered (slope).
In order to more reliably form the cell aggregates one by one in each well 4, the diameter of the opening of the well 4 is larger than the diameter of the bottom of the well, and the inner wall (inner peripheral surface) of the well 4 is tapered. It is desirable that the well be a (slope).

The surface state of at least the bottom surface of the well 4 of the cell culture carrier 1 also has a mean square roughness Rq of 100 nm to 280 nm and a linear density per 1 μm length, similar to the surface roughness of the top surface of the bottom wall 2. It is preferable that it is 1.6 or more and 3.0 or less.
If at least the bottom surface of the well 4 is also in the above-described surface state, the cells are easily maintained in a spherical shape, and cell aggregates are more easily formed in the space in the well 4.
On the other hand, when the root mean square roughness Rq and the linear density are out of the above numerical ranges, the cells adhere to the bottom surface of the well 4 in a flat shape, and cell aggregates are hardly formed.
In the well 4, not only the bottom surface but also the side wall portion (inner peripheral surface) of the well 4 has the same root mean square roughness Rq and linear density as the top surface of the bottom wall portion 2 of the cell culture carrier 1. More preferably, it is within each numerical range.

The inner peripheral surface 3a of the side wall 3 is also more preferably in a surface state in which the mean square roughness Rq is 100 nm or more and 280 nm or less and the linear density per 1 μm length is 1.6 or more and 3.0 or less. .
Accordingly, the mesenchymal stem cells can be more smoothly and reliably moved to the well 4 entering the side wall 3 or to the well 4 closer to the side wall 3 without attaching to the side wall 3. it can.

The plurality of wells 4 formed on the upper surface of the bottom wall portion 2 are preferably arranged in a planar hexagonal lattice pattern as shown in FIG.
The plurality of wells 4 may not be in the form of a plane hexagonal lattice on the upper surface of the bottom wall portion 2 but may be simply a tetragonal lattice arranged at equal intervals in the vertical and horizontal directions. The distance between the adjacent wells 4 becomes substantially equal, and the uniformity of the size of the formed cells can be improved.

Further, as shown in FIG. 3, in a plurality of wells 4 formed on the upper surface of the bottom wall portion 2, between the outer peripheral lines of adjacent wells 4 (between inner peripheral surfaces), a center line L2 connecting the centers O2 of the wells 4 The distance dimension d3 is 10 μm to 50 μm.
When the distance dimension is less than 10 μm, the strength between the wells 4 is insufficient, and the wells 4 cannot be arranged. On the other hand, when the distance dimension exceeds 50 μm, it is difficult to move the spheroidized cells into the well 4, and the mesenchymal stem cells do not completely enter the well 4 and adhere to the bottom wall 2, which is not preferable. .
Even when the plurality of wells 4 arranged in the bottom wall portion 2 are not arranged in the form of a plane hexagonal lattice but in the form of a tetragonal lattice arranged at equal intervals in the vertical and horizontal directions (not shown), The distance dimension d3 (the distance dimension d3 on the center line L2 connecting the centers O2 of the wells 4 between the outer peripheral lines (between inner peripheral surfaces) of adjacent wells) is formed in the range of 10 μm to 50 μm.

Further, at least one (three in FIG. 1) well 4 is provided on the boundary line 6 between the inner peripheral surface 3a of the side wall 3 and the upper surface of the bottom wall 2.
By providing at least one well 4 on the boundary line 6 in this way, the ring-shaped cell aggregate is divided or prevented, and the formation of the ring-shaped cell aggregate is further suppressed.

Here, as shown in FIG. 4, the well 4 formed on the boundary line 6 is such that the inner peripheral surface (opening outer peripheral line) of the well 4 is the bottom wall portion 2 and the side wall portion 3 (inner peripheral portion). It includes a well 4 arranged at a position in contact with the boundary line 6 with the surface 3a).
Thus, also when the well 4 is disposed at a position in contact with the boundary line 6, the cell aggregate is divided by the well 4, and the ring-shaped cell aggregate is suppressed.

In addition, as shown in FIG. 5, the well 4 formed on the boundary line 6 straddles the boundary line 6 and the well 4 in which the inner peripheral surface (opening outer peripheral line) of the well 4 enters the side wall 3. Is included.
Even when the well 4 enters a predetermined dimension into the side wall portion 3, the cell aggregate is divided by the well 4, and the ring-shaped cell aggregate can be suppressed.

Here, the horizontal dimension D of the side wall part 3 preferably has a length that is at least twice as long as the intrusion dimension (intrusion amount d1) of the outer peripheral line of the well 4 into the side wall part 3.
The amount d1 of the well 4 entering the side wall 3 is determined from the intersection of the line L1 connecting the center O1 of the bottom wall 2 and the center O2 of the well 4 and the boundary line 6 with the center O1 of the bottom wall 2 and the well. 4 is the distance to the outer peripheral line (inner peripheral surface) of the well 4 that enters the side wall 3 on the line connecting the centers O2 of the four.
Moreover, the horizontal dimension D of the side wall part 3 means the width dimension in the horizontal direction of an inclined part, as shown in FIG.5 (b).

Here, when the horizontal dimension D of the side wall part 3 is less than twice the intrusion dimension (intrusion amount d1) into the side wall part 3, cells in the cell turbid solution are contained in the well 4 surrounded by the side wall part. The cell aggregates in the wells 4 are less likely to gather, and the cell aggregates in the other wells 4 are smaller than those in the other wells 4, and uniform cell aggregates are difficult to generate.
When the well 4 is positioned on the upper surface 3b of the side wall 3 as shown in FIG. 6A, the medium containing mesenchymal stem cells may drown from the side wall at the time of cell seeding. This is not preferable because cell aggregates having a uniform size cannot be formed in the well 4.
In addition, as shown in FIG. 6B, the well 4 straddles the boundary line 6 and the inner peripheral surface of the well 4 that has entered the side wall 3 has an inclined surface whose angle is substantially the same as the inclination angle of the side wall 3. Even when the inner peripheral surface of the well 4 is continuously connected to the inner peripheral surface of the side wall 3, the internal shape of the well 4 is different from the internal shape of the other wells 4. Thus, when the internal shape of the well 4 is different from the internal shape of the other wells 4, it is difficult to obtain a uniform cell aggregate.

Further, the well 4 disposed on the boundary line 6 is disposed at the intersection of the plurality of straight lines 7 passing through the center of the upper surface of the bottom wall portion 2 and the boundary line 6 as shown in FIG. 7 (plan view). It is preferable that at least six wells 4 are formed on the boundary line between the side wall 3 and the bottom wall 2.
Each center O2 of the well 4 is disposed on an intersection of a plurality of straight lines 7 passing through the center of the bottom wall portion 2 and a boundary line 6 between the side wall portion 3 and the bottom wall portion 2, and the boundary An angle θ2 formed by the straight line passing through the center O2 of two wells 7 adjacent on the line 6 is set to 60 ° or less.

  In this case, at least six wells 4 are provided on the boundary line 6, and cells existing along the boundary line 6 can easily move to the wells 6 provided on the boundary line 6. For this reason, the formation of ring-shaped cell aggregates in the vicinity of the boundary line 6 can be reliably suppressed, and the uniformity of the size of the cell aggregates formed in the well 4 can be improved.

  In FIG. 7, except for the boundary line 6, the illustration of the plurality of wells 4 formed on the upper surface of the bottom wall portion 2 is omitted. The plurality of wells 4 are formed so that the distance dimension on the center line connecting the centers of the wells 4 from the outer periphery of the opening of one adjacent well 4 to the opening of the other well 4 is 10 μm or more and 50 μm or less. Has been.

When culturing using the cell culture carrier 1 formed in this way, it is placed in a plastic case 10 as shown in FIG.
Thereafter, as shown in FIG. 9, a first culture solution (medium) 11 for culturing mesenchymal stem cells is placed in the case 10 and allowed to stand for a predetermined time. When allowed to stand for a predetermined time, as shown in FIG. 10, the first culture solution (medium) 11 enters the pores of the cell culture carrier 1 and reaches the height of the opening of the well 4 by capillary action.

  Although the kind of this 1st culture solution (medium) 11 is not specifically limited, For culture | cultivation of a mesenchymal stem cell, MEM, (alpha) -MEM, DMEM, an eagle culture medium etc. are preferable, for example. Furthermore, substances necessary for maintaining cells such as FBS (bovine serum) and antibiotics are added to this medium. In addition, when using mesenchymal stem cells as a cell therapy tool, it is more preferable to use a commercially available serum-free medium.

Next, as shown in FIG. 11, a second culture solution 12 containing undifferentiated mesenchymal stem cells C and nutrients of the mesenchymal stem cells is dropped on the upper surface of the bottom wall portion 2, and the mesenchymal stem cells C And soak for a predetermined time.
Thus, mesenchymal stem cells C suspended in the culture medium 12 are dropped onto the top surface of the carrier 1 and seeded, so that the mesenchymal stem cells C are not loaded as shown in FIG. And can be settled on the bottom wall portion 2 of the cell culture carrier 1, and smooth aggregation in the well 4 can be achieved.

The number of mesenchymal stem cells seeded on the cell culture carrier 1 is preferably 1 × 10 ⁴ or more and 1 × 10 ⁶ or less per 1 cm ² of the carrier.
By seeding mesenchymal stem cells C at such a density, a three-dimensional cell aggregate having a desired size can be more efficiently cultured using the cell culture carrier 1.

And as shown in FIG. 13, the 1st culture solution (medium) 11 for culture | cultivating a mesenchymal stem cell is further put in case 10, and it leaves still for a predetermined time.
As a result, the mesenchymal stem cells C deposited on the bottom wall 2 of the cell culture carrier 1 fall into the well 4 as shown in FIG. Aggregates are formed.

  That is, in the seeding step by dropping the second culture solution 11 described above, cells adhere to every part of the surface of the carrier 1 regardless of the inside or outside of the well. However, in the step shown in FIG. By immersing the entire cell culture carrier 1 in the liquid 11 and leaving it preferably for 24 hours to 72 hours, the cells attached to the upper surface of the bottom wall 2 outside the well 4 as shown in FIG. It moves into the well 4 without leaving the cell culture carrier 1 and forms a cell aggregate in the well 4.

Then, as shown in FIG. 15, after the culture solution in the case 10 and the cell culture carrier 1 is sucked by an aspirator (not shown), the cell culture carrier 1 in the case 10 is put in the case 10 as shown in FIG. A third culture solution 13 containing a low-molecular compound or protein that induces differentiation of mesenchymal stem cells C into cartilage is added.
As described above, differentiation induction from mesenchymal stem cells C to chondrocytes can be performed in the well 4 by changing the culture medium while maintaining the cell culture carrier 1 used for culturing the mesenchymal stem cells C. it can.

Then, as shown in FIG. 17, the aggregated mesenchymal stem cells C differentiate into chondrocytes 14.
The third culture solution 13 is a medium for inducing differentiation of the aggregated mesenchymal stem cells into tissue cells such as hyaline chondrocytes, adipocytes, osteoblasts, etc., and DMEM is used as the basic medium. Can be used as appropriate. Needless to say, differentiation-inducing media for inducing cartilage, fat, and osteoblasts from mesenchymal stem cells are commercially available.

  Finally, as shown in FIG. 18, after discharging the third culture solution 13, the chondrocytes 14 are taken out from each well 4 and collected.

As described above, according to the embodiment of the present embodiment, mesenchymal stem cells can be aggregated in a uniform shape, and cell aggregates with a uniform differentiation state can be obtained with high probability.
Specifically, since the cell culture carrier is formed in a container shape, there is no gap generated when the plastic container and the carrier are formed separately as in the prior art. It can be made uniform. Moreover, since it is comprised from the ceramic porous body of a predetermined | prescribed average pore diameter and a predetermined | prescribed porosity, the cultured cell can be aggregated in three dimensions, without mixing ceramic particles. Furthermore, since the inner peripheral surface of the side wall portion is formed so as to expand toward the upper side with a predetermined angle of inclination with respect to the upper surface of the bottom wall portion, a mesenchymal stem cell cluster is formed in a ring shape. Can be suppressed.

In addition, since the upper surface of the bottom wall portion is formed in a predetermined surface state, flattened mesenchymal stem cells do not adhere to the upper surface, maintain a spherical shape, and finally enter the nearest well Can do.
Further, the openings of the plurality of wells have a circular shape having a uniform diameter, the diameter of the bottom of the well is formed in a predetermined dimension, and the cross-sectional shape of the bottom of the well has a curved shape that is concavely curved. Since it is formed, mesenchymal stem cell aggregates can be made more uniform in size, and mesenchymal stem cell aggregates with high homogeneity of each cell can be obtained.

Further, at least one of the plurality of wells is formed at a position in contact with a boundary line between the bottom wall part and the side wall part, or straddles the boundary line, and a horizontal dimension of the side wall part is the side wall. Since it has a length that is twice or more the penetration dimension of the well into the part, cells in the vicinity of the boundary line between the bottom wall part and the side wall part can be dropped into the well, Formation of leaf stem cell aggregates can be suppressed.
In addition, since the distance dimension on the center line connecting the centers of the wells between the outer peripheral lines of adjacent well openings is formed to a predetermined length, almost all mesenchymal stem cells are adhered to the bottom wall. Instead, it can enter the inside of the well.

  In addition, at least six wells are formed on the boundary line between the side wall portion and the bottom wall portion, and each of the at least six wells has a plurality of straight lines passing through the center of the upper surface of the bottom wall portion, and The angle formed by the straight line passing through the center of two wells adjacent to each other on the boundary line, which is disposed on the intersection of the boundary line between the side wall part and the bottom wall part, is 60 ° or less. It is possible to more reliably suppress the formation of a ring-shaped cell aggregate that occurs in the vicinity of the boundary line between the portion and the bottom wall portion. The plurality of wells formed on the upper surface of the bottom wall portion are arranged in a planar hexagonal lattice, so that the distances between all adjacent wells are substantially equal, and the size of the formed cells Can improve the uniformity.

  The cell culture carrier according to the present invention will be further described based on examples. In this example, the cell culture carrier shown in the above embodiment was manufactured, tested, and verified.

[Experiment 1]
In Experiment 1, the cell culture carrier having the shape (container shape) shown in FIG. 1 was applied to the inner peripheral surface of the side wall portion with respect to the upper surface of the bottom wall portion by the manufacturing method disclosed in Japanese Patent Application Laid-Open No. 2012-50426 (Patent Document 1). Manufactured with a zirconia ceramic sintered body under the condition of the inclination angle and the number of wells straddling the boundary line between the bottom wall portion and the side wall portion.
In addition, the average pore diameter, porosity, and 2 of the upper surface of the bottom wall portion in the case of a container having a bottom wall portion and a side wall portion having a plurality of wells as a base material of the cell culture carrier of the present invention. The control of the mean roughness Rq, the linear density per 1 μm length, the shape of the well (dimensions, spacing), the horizontal dimension of the side wall, and the taper angle (inclination angle) is controlled by the blending grain size of the ceramic raw material, casting Conditions (mold material, shape, etc.) and drying / firing conditions (temperature, time, atmosphere, etc.) were appropriately selected and combined.
Specifically, the mean square roughness Rq of the upper surface of the bottom wall portion, the linear density per 1 μm length, the average pore diameter of the ceramic sintered body, the porosity, the well opening diameter, the depth, the inside of adjacent wells Table 1 shows the distance dimension on the center line connecting the centers of the wells between the peripheral surfaces. The shape of the bottom of the well was the shape shown in FIG. 2A, and the number of wells was 200 in Examples 1-4, 2500 in Example 5, and 125 in Example 6. Further, the wells straddling the boundary line between the bottom wall part and the side wall part are arranged so that the center of the well is located on the boundary line between the bottom wall part and the side wall part. Further, the horizontal dimension of the side wall is set to 1.5 to 2.5 times the dimension of the perimeter of the well opening into the side wall. In Example 6, the well was disposed such that the outer peripheral line (inner peripheral surface) of the well was in contact with the boundary line between the bottom wall part and the side wall part.

First, the cell culture carrier obtained as described above is placed in a plastic case as shown in FIG.
Then, as shown in FIG. 9, the 1st culture solution (medium) for culture | cultivating a mesenchymal stem cell is put in a case, and it leaves still at 37 degreeC for 6 hours. MSCGM-CD, Bullet Kit (Lonza) was used as the first culture solution (medium).

Next, as shown in FIG. 11, MSCGM-CD, BulletKit was added to a cell culture carrier as a second culture solution containing human mesenchymal stem cells (4 × 10 ⁵ cells) at 37 ° C., 5% The mixture was allowed to stand for 6 hours under CO ₂ and cultured.
The state at this time is shown in FIG. FIG. 19 is a photograph focusing on the bottom wall 2. It was confirmed that the human mesenchymal stem cells seeded on the cell culture carrier settled after standing for 6 hours in culture and adhered to the inside and outside of the well.

And the 1st culture solution was put in the case so that a cell culture carrier upper surface might be covered, and it left still for 72 hours. Thereafter, cell aggregates aggregated in the wells are formed. The state at this time is shown in FIGS. 20 is a photograph focused on the bottom wall, and FIG. 21 is a photograph focused on the well.
Human mesenchymal stem cells seeded on a cell culture carrier move into the well within 72 hours after the addition of the medium, and the cells adhering to the outside of the well (bottom wall) disappear, and cell aggregates are formed at the bottom of the well. It was confirmed to form.

Thereafter, as shown in FIG. 15, after the culture solution in the case and in the cell culture carrier is sucked with an aspirator, the mesenchymal stem cells are induced to differentiate into cartilage in the cell culture carrier in the case as shown in FIG. A third culture solution containing a low molecular weight compound or protein is added. As a third culture solution, DGF (cartilage differentiation induction medium) containing 10 ng / ml TGFβ-3, 100 nM DEX, 50 μg / ml ascorbic acid, 40 μg / ml proline, ITS and pyruvic acid, 37 ° C., The cells were cultured for 21 days under conditions of 5% CO ₂ .

Then, as shown in FIGS. 17 and 18, the aggregated mesenchymal stem cells 13 differentiate into chondrocytes 15. The state at this time is shown in FIG. FIG. 22 is a photograph focusing on the well.
It was confirmed that a cartilage aggregate having a uniform shape was formed in the well of the cell culture carrier.

  Then, after fixing the cells and staining with safranin 0, the cells were observed with a microscope, and the achievement rate of the uniformity of the size of the cells formed in each well was verified. The results are shown in Table 1. Here, the achievement rate means that when a mesenchymal stem cell is seeded on 30 cell culture carriers of each shape and then induced to differentiate into chondrocytes, cell aggregates of uniform size in the wells arranged on the bottom wall of each carrier Is the proportion of cell culture carriers in which wells are formed at 80% or more. The achievement rates in Table 1 are entered by rounding off one decimal place.

  In Comparative Example 1, the inclination angle of the inner peripheral surface of the side wall of the container-shaped cell culture carrier is 90 °, and the ring-shaped cell aggregate described in the above-mentioned problems of the prior art is formed. The phenomenon of differentiation into chondrocytes (see FIG. 24, FIG. 25 to FIG. 29) occurred, and the achievement rate was 33%, which was a very low value.

  Further, as shown in Table 1, the inclination angle of the inner peripheral surface of the side wall portion is 100 ° to 135 °, the number of wells in contact with the boundary line between the bottom wall portion and the side wall portion is one or more, or the bottom wall portion A good achievement rate (achievement rate of 70% or more) could be obtained in the range where the number of wells straddling the boundary line between and the side wall portion was 1 or more. Furthermore, a good achievement rate (achievement rate of 90% or more) can be obtained when the central angle of the arc of the adjacent well on the boundary line between the inner peripheral surface of the side wall portion and the upper surface of the bottom wall portion is 60 ° or less. did it.

[Experiment 2]
In Experiment 2, as Comparative Example 2, the horizontal dimension D of the side wall was set to 1.5 times the dimension d1 of the well entering the side wall. The conditions were the same as in Example 1.
In this experiment, Comparative Example 2 is less than Example 1 in that the cell aggregates in the wells that entered the side walls are smaller than the cell aggregates in the other wells, and it is difficult to generate uniform cell aggregates. It was recognized that
Therefore, it was found that the horizontal dimension D of the side wall part is preferably at least twice the dimension d1 of the well entering the side wall part.

  From the results of Experiments 1 and 2 above, according to the cell culture carrier of the present invention, mesenchymal stem cells can be aggregated in a uniform shape, and cell aggregates with a uniform differentiation state can be obtained with high probability. Was confirmed.

DESCRIPTION OF SYMBOLS 1 Cell culture support | carrier 2 Bottom wall part 3 Side wall part 3a Inner peripheral surface 4 Well 6 Boundary line 10 Case 11 1st culture solution (medium)
12 Second culture medium (medium)
13 Third culture solution (medium)
14 Chondrocytes θ1 Inclination angle d1 Well penetration into the side wall

Claims (3)

A cell culture used for culturing a mesenchymal stem cell, which is formed in a container shape having a bottom wall portion and a side wall portion provided at the periphery of the bottom wall portion, and in which a plurality of wells are formed in the bottom wall portion A carrier,
An average pore diameter is 0.1 μm or more and 0.6 μm or less, and a porous ceramic body having a porosity of 40% or more and 55% or less,
The inner peripheral surface of the side wall portion is formed so as to expand as it goes upward with an inclination angle of 100 ° to 135 ° with respect to the upper surface of the bottom wall portion, and the mean square of the upper surface of the bottom wall portion The roughness Rq is 100 nm or more and 280 nm or less, and the linear density per 1 μm length is 1.6 or more and 3.0 or less,
The openings of the plurality of wells have a circular shape having a uniform diameter, and the bottom of the well has a diameter of 100 μm or more and 550 μm or less, and the cross-sectional shape of the bottom of the well is formed into a curved shape that is concavely curved.
At least one well of the plurality of wells is formed at a position in contact with a boundary line between the bottom wall part and the side wall part, or straddles the boundary line, and a horizontal dimension of the side wall part extends to the side wall part. Having a length of at least twice the penetration size of the well,
And the distance dimension on the center line connecting the centers of the wells from the outer periphery of the opening of one adjacent well to the outer periphery of the opening of another well is 10 μm or more and 50 μm or less. Carrier. The upper surface of the bottom wall part surrounded by the side wall part is circular,
At least six wells are formed on a boundary line between the side wall and the bottom wall,
The center of each of the at least six wells is disposed on an intersection of a straight line passing through the center of the upper surface of the bottom wall portion and a boundary line between the side wall portion and the bottom wall portion,
The cell culture carrier according to claim 1, wherein an angle formed by the straight line passing through the centers of two wells adjacent to each other on the boundary line is 60 ° or less.   The cell culture carrier according to claim 1 or 2, wherein the plurality of wells formed on the upper surface of the bottom wall portion are arranged in a planar hexagonal lattice pattern.