e) Step of suspending the ลูกกลม in the อาหารเลี้ยง
30 After each spheroid is grown to a desired size, the cells cultured in each recess 11 are
suspended in the อาหารเลี้ยง by agitating the อาหารเลี้ยง in each ตู้เพาะเลี้ยง 1. For example, this step is carried out by agitating the อาหารเลี้ยง. Specifically, the agitation of the อาหารเลี้ยง can be done by any one of the following means: (1) agitation of the อาหารเลี้ยง by shaking each ตู้เพาะเลี้ยง 1; (2) agitation of the อาหารเลี้ยง by sucking and discharging the อาหารเลี้ยง (pipetting); (3)
13
agitation of the อาหารเลี้ยง by disposing a stirring blade in each ตู้เพาะเลี้ยง 1; (4) agitation of the อาหารเลี้ยง by placing a stirrer in each ตู้เพาะเลี้ยง 1; and (5) agitation of the อาหารเลี้ยง by a combination of two or more of the above-mentioned means (1) to (4).
f) Step of recovering the cells
5 The อาหารเลี้ยง including the cells in each ตู้เพาะเลี้ยง 1 is sucked by a suction
machine, to thereby recover the cells (ลูกกลม) suspended in the อาหารเลี้ยง.
[0028]
As described above in the first embodiment, seeding of cells, การแทนที่ of a อาหารเลี้ยง,
and harvesting of cells can be performed in the same chamber, and in addition, ลูกกลม can be 10 recovered from each ตู้เพาะเลี้ยง.
Culture of cells using the ตู้เพาะเลี้ยงs 1 of the first embodiment enables formation of a spheroid having a desired size on the ส่วนล่าง 11. Further, the cultured ลูกกลม can be efficiently recovered. Specifically, the structure of each recess 10 including the bottom
portion 11 and the ส่วนช่องเปิด 12 makes it possible to easily maintain a state in which cells 15 adhering to the ส่วนล่าง 11 or being suspended in the อาหารเลี้ยง are prevented from being
removed when the อาหารเลี้ยง is sucked during การแทนที่ of the อาหารเลี้ยง. Thus, it can be
expected that the removal of cells from the ส่วนล่าง 11 is suppressed. On the other hand,
during the harvesting of cells, when the อาหารเลี้ยง in the ส่วนล่าง 11 is sucked and
discharged, it can be expected that the อาหารเลี้ยง is allowed to easily flow through the opening 20 portion 12. It can also be expected that the use of the hemispherical shape of the ส่วนล่าง
11 contributes to the formation of ลูกกลม with a uniform shape and size. [0029]
Second Embodiment
While an example of the structure in which the ส่วนล่าง 11 has a hemispherical 25 shape has been described in the first embodiment, other shapes of the ส่วนล่าง will be
described in a second embodiment. The ส่วนล่าง may have any form, such as a shape
formed using a part of a spherical shape, a truncated cone shape, or a linear shape. The linear
shape of the ส่วนล่าง is a form having no substantial ส่วนล่าง and having a recess
formed only by an ส่วนช่องเปิด. Figs. 4 to 7 show examples of the shape of each recess
30 according to this embodiment. Figs. 4 to 7 show โพรง 20A to 20D having ส่วนล่างs
21A to 21D, respectively, which are different from the ส่วนล่าง 11 of the first embodiment.
Since the ส่วนช่องเปิด 12 can be formed with the same shape as that of the first embodiment,
Figs. 4 to 7 show examples of the shape of each recess in which the ส่วนช่องเปิดs having the
same shape are combined.
While in the first embodiment, a hemispherical shape, which is a shape obtained by
dividing a spherical shape into halves, is used for the ส่วนล่าง 11, Figs. 4 and 5 show
examples in which different hemispherical shapes are used for the ส่วนล่าง. Fig. 4 shows 5 the ส่วนล่าง 21A for which a portion less than a half of a spherical shape is used. In other
words, Fig. 4 shows a case where a part of a hemispherical shape is used for the ส่วนล่าง
21A. Fig. 5 shows the ส่วนล่าง 21B having a cylindrical shape with a hemispherical
bottom. In the case of the shape of the ส่วนล่าง 21B shown in Fig. 5, as the length of the
cylindrical portion increases, the cells are less likely to be suspended into the อาหารเลี้ยง from the 10 ส่วนล่าง 21B during harvesting of the cells. Accordingly, it is preferable to adjust the
length of the cylindrical portion. For example, it is preferable to form the ส่วนล่าง 21B and the ส่วนช่องเปิด 12 so as to maintain the same ratio (1:1) between the depth (height) of the ส่วนล่าง 21B and the depth (height) of the ส่วนช่องเปิด 12.
Fig. 6 shows the ส่วนล่าง 21C for which a truncated cone shape is used. When 15 the ส่วนล่าง is flat, the reflection and interference of light can be reduced, and thus it is
useful for observation with a microscope.
Fig. 7 shows an example of the shape of the recess 20D in which the ส่วนล่าง
21D has a linear shape, i.e., the ส่วนล่าง 21D does not fowl a ที่ว่าง. The efficiency of
culture and harvesting of cells in the recess 20D is lower than that of ตู้เพาะเลี้ยงs having 20 other shapes. However, the recess 20D has an advantage in facilitating the production process
for the ตู้เพาะเลี้ยงs.
[0031]
A case where the ส่วนช่องเปิด 12 is formed with a shape similar to that of the first embodiment has been described in this embodiment. However, the การประดิษฐ์นี้ is not
25 limited to this case.
The method of culturing cells using the ตู้เพาะเลี้ยงs according to this embodiment is similar to that of the first embodiment, and thus the description thereof is omitted.
The ตู้เพาะเลี้ยงs according to this embodiment can provide the same advantageous effects as those of the first embodiment.
30 [0032]
Third Embodiment
A mode in which the shape of the ส่วนช่องเปิด 12 is a circular shape or a
substantially circular shape has been described in the above embodiments. A ตู้เพาะเลี้ยง including ส่วนช่องเปิดs each having a shape other than a circular shape or a substantially
15
circular shape will be described. An end of each ส่วนช่องเปิด may have a shape other than a
circular shape or a substantially circular shape, such as a hemispherical shape, a trapezoidal
shape, or an inverted triangular shape. On the other hand, it is necessary that the shape of the
boundary where the ส่วนช่องเปิด contacts the ส่วนล่าง (the boundary portion of the 5 ส่วนช่องเปิด) be the same as the shape of the boundary portion of the ส่วนล่าง. Figs. 8
and 9 show โพรง 30A and 30B, respectively, each having an end with a shape different from
that of the ส่วนช่องเปิด 12 of the first embodiment. While Figs. 8 and 9 show the same
ส่วนล่าง 11 as that of the first embodiment, a combination of any of the ส่วนล่างs
21A to 21D of the second embodiment, or a ส่วนล่าง having another shape may be used. 10 The ส่วนล่าง and the ส่วนช่องเปิด may have any shape as long as an inclined surface
can be formed continuously at the boundary between the ส่วนล่าง and the ส่วนช่องเปิด. [0033]
Fig. 8 shows an example of the shape of an end of the ส่วนช่องเปิด 32A that is
formed in a curve. Fig. 8 is a top view of the recess 30A. An end of the ส่วนล่าง 11 is 15 indicated by a circle having the equivalent diameter R, and the outer periphery of the opening
portion 32A is indicated by a curve. The end of the ส่วนช่องเปิด 32A has a curved shape
which is not symmetric in the horizontal direction and the vertical direction. However, the end
of the ส่วนช่องเปิด 32A may have a shape which is symmetric in the horizontal direction or
the vertical direction. Fig. 9 shows an example in which an end of the ส่วนช่องเปิด 32B has a 20 rectangular shape. Although Fig. 9 shows an example in which an end of the ส่วนช่องเปิด
has a square shape, the end of the ส่วนช่องเปิด may have another polygonal shape, or a
combination of a curve and a straight line. Fig. 9 is a top view of the recess 30B. The end of the
ส่วนล่าง 11 is indicated by a circle having the equivalent diameter R, and the outer
periphery of the ส่วนช่องเปิด 32B is indicated by a solid square. For example, the shape of 25 the end of the ส่วนล่าง may be modified so as to adjust the area of the ที่ว่าง between the
end of the ส่วนล่าง and the adjacent recess. Since it is necessary for the shape of the end
of the ส่วนช่องเปิด to play a role of promoting the suspension of cells, the taper angle is
important.
In the shape examples shown in Figs. 8 and 9, the taper angle has a value that varies
30 depending on the shape of the ส่วนช่องเปิดs 32A and 32B. This is because the inclination of
the inclined surface that forms the wall varies depending on the shape of the ส่วนช่องเปิดs 32A and 32B.
[0034]
Each of the shapes of the ส่วนช่องเปิดs illustrated in this embodiment can be
16
combined with the shape of the ส่วนล่าง 11 described in the first embodiment, or the
shape of the ส่วนล่าง described in the second embodiment. In addition, these shapes can also be combined with a shape other than the shapes of the ส่วนล่าง illustrated in the above embodiments, as a matter of course.
5 The method of culturing cells using the ตู้เพาะเลี้ยงs according to this embodiment
is similar to that of the first embodiment, and thus the descripti