1
Description
Title of the Invention: CULTURE SUBSTRATE AND CULTURE SHEET
5 Technical Field
[0001]
The present invention relates to a technique of
culturing ani~al and plant cells using a culture substrate,
and graphically forming spheroids (3D tissues), and
10 monolayer tissues (2D tissues) of the cells.
Background Art
[0002]
In the process of developing pharmaceuticals, in
15 vitro assays using cells instead of animal experiments have
been required. In particular, the demand for applying such
in vitro assays to the screening and toxicity and
metabolism tests of candidate pharmaceutical substances has
been increasing.
20 [0003]
In such a background, approaches of alternative.
methods using cells in place of conventional animal
experiments has been actively attempted, but many of such
approaches have limitations in predicting their clinical
25 reactions. This assumedly because the forms of the cells
2
tit are not mimicking their actual in vivo structures in these
culture methods (Non-Patent Literature 1). Therefore, the
construction of 3D tissues which exhibits functions more
similar to those of living bodies has been attempted so far,
5 and 3D tissues has been successfully formed for various
cell strains.
[0004)
As a substrate for forming 3D tissues of cells, a
sheet (nanopillar sheet) for culture in which regularly
10 arranged ultrafine pillar structures or protrusions are
formed on the surface of a sheet has been developed, the 3D
tissues formed has the problems that they have high release
properties from the substrate (Patent Literature 1), and
that they are lost in the process of medium change.
15 Moreover, since it is impossible to control the diameter of
formed 3D tissues, it entails the problem that their sizes
are not uniform, and therefore the performance of each of
the 3D tissues is varied. It is thus still premature as a
practical formation method.
20 [0005)
To this end, a technique of providing minute cavity
structures in a culture substrate, and forming a single 3D
tissue per cavity (cellular tissue microchip) has been
developed so far (Patent Literature 2, Non-Patent
25 Literature 2). A feature of this technique is that by
3
It applying a substance having adhesion to a predetermined
region around the center of at the bottom of the cavity, a
cell adhesive region and a cell non-adhesive region are
defined, and the cavity itself is rotat~d by a rotation
5 drive apparatus or the like to perform rotation culture, so
that cultured cells are retained around the center of the
bottom of the cavity which is the cell adhesive region.
Citation List
10 Patent Literature
[0006]
Patent Literature 1: Japanese Unexamined Patent Application
Publication No. 2005-312343
Patent Literature 2: Japanese Unexamined Patent Application
15 Publication No. 2006-121991
Non-Patent Literature
[0007J
Non-Patent Literature 1: ~The Use of 3-D Cultures for High20
Throughput Screening: The Multicellular Spheroid
Model"Leoni A. Kunz-Schughart, James P. Freyer, Ferdinand
Hofstaedter, and Reinhard Ebner J Biomol Screen, 9: 273-285
(2004)
It N~~~~~WG ~~~§u~ ~~~, +~~~?R4+q~ f?QQ~)
Non-Patent Literature 3: ~Formation of Hepatocyte Spheroids
with Structural Polarity and Functional Bile Canaliculi
Using Nanopillar Sheets." R Takahashi, H Sonoda, Y Tabata
5 and A Hisada, Tissue Eng Part A, 1-45 (Mar 4, 2010)
Summary of Invention
Technical Problem
[00081
10 While cellular tissue microchips have such features,
in order to compulsorily adhere cells onto specific
portions on the surface of the substrate, the cell adhesive
region and cell non-adhesive region need to be defined by
applying a chemically synthesized substance on the surface
15 of the sUbstrate, which entails some problems.
[0009J
First, these chemicals applied may adversely affect
the growth of cells, but also this operation requires
application or adhesion of chemicals in the hyperfine
20 region, which greatly complicates the operation and
requires production costs.
[0010]
Moreover, when inoculated cells fall into nonadhesive
regions, they are inevitably disposed of along
25 with the medium when the medium is changed during culture,
5
tt which is hardly considered as an efficient culture method.
Furthermore, it is suspected that the cells which have
fallen into the adhesion region are caused to form tissues
compulsorily by rotation culture, and therefore stress is
5 exerted on cells, which leads to a lowered activity.
[0011]
Meanwhile, known nanopillar sheets also have the
problems that it is difficult to control the cell movement
on the substrate plane, and that it is impossible to
10 control the dimension and diameter of the 3D tissues formed.
At the same time, it also has the problem that it is
impossible to retain the formed 3D tissues in a target
position.
[0012]
15 An object of the present invention is to provide a
culture sheet, a culture substrate, and a cell culture
method using the same which enable forming 3D tissues
having a uniform diameter without applying chemicals on the
surface of the culture substrate, and further retaining the
20 3D tissues in a target position.
Solution to Problem
[0013]
In order to achieve the above-mentioned object, the
25 present invention provides a culture substrate and a
5
6
culture sheet in which a culture reg~on is provided, a
plurality of projections are formed in the culture region,
a partition which partitions the culture region and is
taller than the projections around the culture region form,
and the constitutional proportion of the projections in the
culture region is in the range from 20% to 75%.
[0014]
Moreover, in order to achieve the above-mentioned
object, the present invention provides a culture substrate
10 and a culture sheet in which a culture region is provided,
a plurality of projections are formed in the culture region,
a partition which partitions the culture region and is
taller than the projections around the culture region is
formed, and the constitutional proportion of the
15 projections in the culture region is in the range from 40%
to 50%.
Advantageous Effe<;::ts of Invention
[0015]
20 By applying the present invention, formation of 3D
tissues can be realized under circumstances with little
stress while using only a single material and maintaining
their activities by promoting cell movement which is a
function inherent to cells.
25 [0016]
~ ~9~~9Y.~~'. py ~nt~g~~~~¥ ~~Q¥ig~D9 q +im~t~Q ~~9.iQn,
i.e., a partition, from the same material, cells inoculated
within the limited region are all involved in the formation
of a single 3D tissue. This achieves a very efficient
5 culture method, and also leads to the expectation that the
sizes of a plurality of 3D tissues formed for the
respective limited regions are uniform and homogenous,
which is effective in cell assays.
[0017]
10
15
furthermore, it is expected that the 3D tissues are
retained in a target position within the limited region,
i.e., the partition. Furthermore, 2D tissues Can be formed
depending on the purpose. Similar effects are also
expeeteq on the 2D tissues.
Brief Description of Drawings
[0018]
[Fig. 1] Fig. 1 is a drawing which shows the culture sheet
according to Example 1 and the hole structure in the
20 culture sheet.
[Fig. 2J Fig. 2 is an enlarged view which shows the
nanopillar structure according to ~xample 1.
(Fiq. 3) Fig. 3 is a drawing which shows a chamber slide
f. according to Example 1, with the culture sheet affixed.
25 thereto.
tt (r!~ __ ~J Etg t ~ ~§ q Q~~~4n~ ~h~gb ~bgW§ t~~ GQ~$t~t~~ip,~
of a plate frame body according to Example 2.
[Fig. 5] Fig. 5 is a drawing for ~llustrating the flow of
the ultrasonic welding ot the plate and culture sheet
5 according to Example 2.
[Fig. 6) Fig. 6 is a drawing which shows the flQwchart of
h~patocyte culture according to Example 3.
[Fig. 7J Fig. 7 is a drawing which shows a photograph of a
hepatocyte 3D tissue by the culture sheet by the hepatocyte
10 culture flow according to Example 3.
[Fig. 8] Fig. 8 is a drawing which shows a two-stage and
mUlti-stage nanopillar culture sheet according to Example 4.
[Fig. 9] Fig. 9 is a drawing which shows the types of
arrangement patterns of the nanopillars of Examples.
15 [Fig. lOA] Fig. lOA is a drawing which shows the results of
the cell culture (the state of cells) when culture sheets
having different pillar diameters shown in Fig. 9 are used.
[Fig. lOBJ Fig. lOB is a drawing which shows the results of
the cell culture (number of cells formed) when culture
20 sheets having different pillar diameters shown in Fig. 9
are used.
[Fig. 11] Fig. 11 is a drawing which shows an inclined
nanopillar culture sheet which is a variant of Example 4 of
culture sheets.
25 [Fig. 12] Fig. 12 is a draWing which shows a well of the
gij~t~+.~ ~b~~t hgy~~g q §ur~gG~ ten§~Qn ~Moigi~g p~tt~Fnt
',' : '.", -- ...' ~ " .. ' " ,- .• '". ". ,-,' .' • '-'.' '.' ". . .• , .,- ,-., • - , '.' , ",', 'r • -. _ <.' ~.,...,. __. ,,' .
which is variant 4 of Example of the culture sheet.
[fig. 13A] Fig. 13A is ~ drawing which shows an appearance
perspective view, top view, upper and lower side view of
5 the culture substrate in Example 1.
[Fig. 13B) Fig. 13Bis a partially enlarged view of the
culture substrate in Example 1, which shows an A-A, B-B
partially enlarged view and a C-C, D-D partially enlarged
view.
10 (Fig. 13C) Fig. 13C is a partially enlarged view and end
view of the culture substrate in Example 1, and is a
drawing which shows an E-E, F-F partially enlarged view,
line G-G end view.
[Fig. 14A] Fig. 14A is a drawing which shows a perspective
15 view and bottom view of the appearance of the culture
substrate in Example 2.
[f~g. 14B] Fig. 14B is a drawing which shows a top view,
upper and lower side view of the CUlture substrate in
Example 2.
20 [Fig. 14C) Fig. 14C is a partially enlarged view and
partial cross-sectional view of the culture substrate in
Example 2, which shows an A-A, B-B partially enlarged view,
a C-C, D-D partially enlarged view, and an H-H crossse~
tional view.
25 [Fig. 14DJ Fig. 14D is a partially enlarged view, and an
tD eng view o~ tbe c~+ture ~~$.t~~t~ i.n ~~arop1~ g" ~oiGb ~hQW$
an E-E, F-F partially enlarged view, and a line G-G end
view.
[Fig. 15A] Fig. 15A is a drawing which shows the culture
5 sheet and the hole structure in the culture sheet according
to Examples 5 and 6.
[Fig. 15B] Fig. 155 is a schematic diagram which shows an
assembly of projection portions having different diameters
according to Examples 5 and 6.
10 [Fig. 15CJ Fig. 15C is a drawing which shows an SEM image
of the culture sheet of the assembly of projectiqn portions
having a diameter of 80 vm according to Examples 5 and 6.
[Fig. 150J Fig. 15D is a drawing which shows an SEM image
of the culture sheet and the assembly of projection
15 portions having a diameter of 20 ~ according to Examples 5
and 6.
[Fig. 16A] Fig. 16A is a drawing which shows an example of
the distance between the center of a hepatocyte 3D tissue
and the center of the hole structure by the culture sheet
20 by the flow of hepatocyte culture according to Example 7.
[Fig. 16B] Fig. 16B is a drawing which shows another
example of the distance between the center of the
hepatocyte 3D tissue and the center of the hole structure
bY the culture sheet by the flow of hepatocyte culture
25 according to Example 7.
tD ~[!~~ ~§QJ ~;9~ +§g ~§ ~ ereW~nq wbAgn §bg~~ ~nQtn~r
example of the distance between the center of the
hepatocyte 3D tissue and the center of the hole structure
by the culture sheet by the flow of hepatocyte culture
5 according to Ex~mple 7.
[Fig. 16D] Fig. 16D is a drawing which shows another
example of the distance between the center of the
hepatocyte 3D tissue and the center of the hole structure
by the culture sheet by the flow of hepatocyte culture
10 according to Example 7.
[r~g. 17] Fig~ 17 i~ a drawin9 whiGh ~h9W~ an e~~mpl~ ot a
I
photograph of the hepatocyte 3D tissue by the culture sheet
by the flow of hepatocyte· culture according to Example 7.
[Fig. 18] Fig. 18 is a drawing which shows an example of a
15 photograph of the hepatocyte 3D tissue by the culture sheet
by the flow of hepatocyte culture.
Description of Embodiments
[0019]
20 The best mode for realizing a method for culturing
c~lls using a culture sheet, and forming the 3D tissues
which is a cell cluster, or 2D tissues will be described
below in detail.
25 Ex~ple 1
12
'~'~
is applied to the chamber slide which is a culture sheet
retaininq member. Hereinafter, a sheet which has a
5 partition structure which forms the culture region in the
present invention on a known nanopillar sheet, and on which
a plurality of projections are formed inside the partition
structure is referred to as a culture sheet.
The culture sheet is formed from a material which has no
10 adverse effect on cells, in this example, it is polystyrene.
However, it goes without saying that the material is not
limited to polystyrene.
[0021]
Fig. 1 is a schematic diag~am of a scanning electron
15 micrograph of a culture sheet 100 prepared in this Example.
Simultaneously, it shows the structure of one of holes 101 .
(hereinafter referred to as hole) constituted by a
plurality of partition structures 102 existing in a single
culture sheet. The inside of the hole 101 constitutes a
20 culture region by cell tissue formation unit.
(0022]
A plurality of projections 102 retained at the bottom
of the ho+e 101 includes a plurality of microprojections
103 (hereinafter also referred to as projections, pillars
25 or nanopillars). Moreover, the diameter of this hole 101
13,
tI ;~ ~ P9+~ ~;~~~~~f ~9~f Ip th~ ~~+~BF~ ~h~~t +QQ" th~ p~~~
i9t ~pg~~q~~9 th~ ~~9,~e~w~~piQP'~g ~~~t.~t.~qn ~~*~ l.Qa ~~g §
plurality of projections 103 formed inside the hole 101 are
formed from the same material integrally. It should be
5 noted that the shape of this hole 101 is not limited to
round, but may have other shapes such as a square shape.
(0023]
In this manner, the hole 101 and the plurality of
projections 103 fermed in$ide the hole 101 including the
10 partition wall 102 are formed integrally as the culture
sheet 100 from a sin~le material which has no adverse
effects on cells, whereby cells can be grown without
foreign substances bonding to the cells in the culture
steps. Furthermore, since cells are grown in each of the
15 Partitions, cells of a homogeneous size can be fOrmed.
(0024]
Moreover, a plurality of projections are provided
within the partition wall 102 arranged in a surrounding
manner, and therefore the cell movement which is the
20 ability inherent to the cells is promoted, and cells are
grown by the movement so that cell culture which can
maintain the cell activity is possible with no influence of
disturbance (stress) by rotation culture or the like.
[0025]
25 When alculture region is to be formed while these
98+~~ l.q~ ~~9 ~+q~~ctiQ~ ~s.~~~l¥ +9~ ~~~ Pf8~~~~9
; . :.-.• "_ . ,; :", .c. >_ '.; - . ;:":' .": ,',-" . '. ,', .. -.
s.ep~r~t~ly, they need to be joined. by ~qhesion or welqing.
[0026]
5 adhesive components enter into the culture region, which
may adversely affect generated cells. In joining by
welding, the inner diameter of the hole 101 is a hyperfine
region diameter on the cell formation level, and therefore
it is very difficult to perform welding while forming a
10 target cell region and not dam~ging the partitions and
projections. When the partitions and projections have
damages and deformations, unwanted stress m~y be applied on
the cells in the process of cell formation, and the
movement of the cells themselves may be impaired.
15 . (0027]
and projections 103 constituting the holes 101 which forms
the culture region are ~referably formed integrally. By
forming integrally in such a manner, it is preferable
20 because culture excluding the influence of unwanted
Gomponents other than those required for cell culture can
be performed.
[002SJ
Subsequently, an enlarged view of the projection 103
25 is shown in Fig. 2. A pillar diameter indicates a diameter
e ~Q~ 9f ~h~ tip Qf trl1~ ~:r:Qject~QI1! -8- B~p,~r p~t.2h ~I?-g:i.G~t€l§
q distance 107 f~om the Qenter of the tip of the p:~ojeGtion
to the c~nte~ 9f th~ tip of the a~jacent projection. A
pilla~ height indicates a height 108 from the tip of a
5 nanopillar to the bottom thereof. Fig. Z{a) and Fig. 2(b)
indicate a square arrangement and a triangle arrangement,
respectively, of nanopillars of this Example.
[0029]
In this Example, culture sheets in which the pillar
10 diameter, pillar pitch and pillar height are 2.0 ~, 4.0 ~,
and 1.0 ~, respectively, were used, but as will be
described later, such culture sheets are not necessarily
used. The height of the partition structure is 70 pm in
this ~xample, but this value is not necessarily used, and
15 suitably the height may be such that the formed cells do
not get over the partition.
[0030 ]
The culture sheet lOO in this Example is produced by
the method described below. A mold in which round holes
20 each haVing a diameter of 200 pm and depth of 70 pm are
arranged in the form of squares, and micropores each having
polystyrene film having a thickness of 400 pm at 135°C and
25 a pressure of 2 MPa. The film was took out from a press
~f9~;n~ ~tt~f ?e;~~ 9Q~+~9 ~? f~?m ~!m~~Pf~Hf~'. ~p'g th~
" .' ,.~ '", ""., ;'_'~, ";'. ',' >~;.,~ :. "'\'-'~"'- -, ,..;'.~' '._ ""C .,:. ,-- '.'
mQ~q W~~ peeled off f~QID the golystyre.g~ film, where.by ~
, - - . .
culture sheet retaining a pluralit.y of holes each having a
hole diameter of 200 ~ and having a plu~ality of
5 projections at the bottom thereof can be produced.
[0031J
Herein, a mold material is silicon wafer, and in
order to prevent adhesion with the polystyrene film during
the production of the culture sheet, a mold releasing
10 process is performed in advance with a fluorine-based mold
releasing agent. Silicon wafer was used as the mold
material in this Example, but a mold made from other metal
mat.erials and the like ~ay be also used.
[0032]
15 As shown in Fig. 3, the culture sheet 100 produeed. by
integral molding from the single material in this manner
was cut into 2-cm square pieces in this example, and a
surgical glue 110 was applied onto the glass bottom of the
chamber slide 109 to adhere the chamber slide 109 and the
,
20 culture sheet 100, whereby the chamber slide 109 with the
cUlture sheet 100 affixed thereto is produced. It should
be noted that in Fig. 3, 109a represents a frame for
partitioning the culture sheets 100. This frame 109a is
formed from, for example, a plastic material or the like.
25 It should be noted that the shape of a frame body such as
t' h'..~<.~.. +"fa"r"o~ ~q~.~," ~~, pg" t +'.~"m.. ~' t~q £9 §gy~~~~ pyt ~~¥ ~~ 9ther .(0" ", ....... '.'. _ ." ~. ,',
shapes such as a round shape.
[OO~3J
Figs. 13 A, 13B, 13C, show the overal+ constitution
5 diagram and principal part cross-sectional view of the
chamber slide with the culture sheet of this Example
affixed thereto.
Fig. 13A is an appearance perspective view, top view, and
upper and lower side views of the culture substrate in this
10 Example. Illustration of left and right side elevational
views is omitted since its form is obvious from the
perspective view.
Fig. 13B is a part~ally enlarged view, whiCh shows an ~~~,
B-B partially enlarged view, and a C-C, D-D partially
15. enlarged view.
Fig. 13C is a partially enlarged view and end view, which
shows an E-E, r-F partially enlarged view, and a line G-G
end view.
[0034)
20 The article shown in Figs. 13A to 13C is a culture
device (culture containers) for CUlturing cells of humans,
animals, plants and others, and are each constituted by the
culture sheet 100 and a retaining member (chamber slide)
109 which retains the culture sheet 100. A plurality of
25 partition portions 102 are formed on the surface of the
5
RH~;ij~' !h'~S !QO, !n~ ~s ~~g~~~I~ It t~, ~Q;~9~ e! ~b'
.' .,. ; C" ... .' • .. ," .. " :--,,,, ~. '.
[OO~5J
f~fth~r~9~~' 9~~t~~~ r~g~9n~ h~V~~9 ~ P+~F~+~ty 9+
~inute projection portions 103 within the partition portion
are formed respectively. When target cells to be cultured
are added to the inside of the hole portion 109a, as added
to the sheet surface forming the culture regions within the
10 partition portion 102, the target cell is retained in the
plurality of minute projection portions 103 and cultured.
E}{a~ple 2
[0036J
1~ S~p~e,~ently, Example 2 will be de$cribed with
reference to Figs. 4 and 5. In Example 2, the constitution
of a JIlultiwell plate w~th a cuJ,ture sheet and a prod~ction
example thereof will pe shown. Fig. 4(a) i$ a bottom view
of a frame body III const:d::uting the multiwell plate. The
20 frame body 111 which is a culture sheet retaining member is
such that has 24 cylindrical hole portions lIla in total,
arranged in 4 rows and 6 columns, fo~ed in an area
measuring about 125 mm in width, about 80 mID in length, and
about 20 mID in height. The material used is polystyrene.
25 [0037J
tt ~S~ B~~f ~f 99~~~ ~R~~g B~ ~~~ ~~~m~ ~9~¥ n~f:~~+~¥'
;~n.~~$ fr~Qrn ~ t9 ~~~~~ va+~~Q g~P~~A~P9 QQ tA~ ~~~! ~pg
tn~refor~ the n~er Qt holes qp this ~rame ~oqy ~s not
l~miteq to 24~ !h~m~terial qf the ftam~ pody is not
5 limited to polystyrene either.
[003e]
In pr04~~~ng t~e culture s~strate, the frame QQq¥
111 and the culture sheet 100 is joined by ultrasonic
welding.
ro [0039]
The following processes are perfo~eq O~ the frame
body 111 in advance. As the first process, a projection
for fixing film 112 is processed at the bottom of the frame
body 111 for the purpose of preventing the cell culture
15 sheet and the plate from being shifted due to the vibration
of ultrasonic waves provided when the frame body 111 and
the culture sneet 100 are welded. As the second process, a
rib structure ~13 ~s prov~ded'to weld the cUlture sh.et ~y
ultrasonic waves~
20 [0040]
Figs. 4(b) and 4(c) are snows the cross-sectional
views at lines B~B' and A~A', respectively, in Fig. 4(a).
Moreover, holes 114 having the same diameter are provided
in the culture sheet in the same position when both are
25 overlapped so that the projection engages with the
§~SS~§~~y~+y! ~h~~ ~f~~ ~Q9X
"'.. .," -'- : ,',' . "" ,'..... - . ,.~ ' ....
a~Q the culture sheet 100 are adherep by ultrasonic welding.
(0041J
The step of the welding is shown in Fig. 5. First,
5 the holes of the projection for fi~ing film of the frame
body and of the qulture sheet are piaGed together and
stacked (Fig. 5(a)). SUbsequently, ultrasonic waves are
produced from the culture sheet side from an ultrasonic
wave oscillator via a converter, a booster, or further a
10 horn, and both are welded (Fig. 5(b)). A horn is an
apparatus for welding by irradiating an appropriate
position with ultrasonic waves of an appropriate energy. A
specific apparatus designed so that ultrasonic waves are
generated appropriately along the position of the rib
15 structure w.as produced and used. 115 shows a top view of
the thus~producep plate.
[0042J
The frame bod¥ and the culture sheet were joined by
using ultrasonic weldinq in this Example, but it goes
~o . without saying that the joining is not limited to this
method. Formation of a plate can be realized without any
intervention of organic matters such as adhesives which
affects cells by ultrasonic welding. Therefore, no adverse
effects are caused on cells. Needless to say, this Example
25 is a culture sheet which is applicable and useful not only
5
21,
~Sl ~9~~g!fY ~It9 W~1;eBQt~§~ ~~~.~~ tV: Q~~ ~~H~ 9~~~;~R~~Qt
. '~.' '{:~. './ " .'.' , - ,
processes, but ~lso to the formation of organizations
It is nee.ctle.ssto say that bY provid.ing a plur~lity
of the rib structures at the bottom of the frame 109a also
in the culture substrate of the chamber slide shape shown
as an example in Example 1, and performing welding with the
culture sheet 100 by the rib structures, the culture
10 substrate can be produced by a joining method similar to
this Example.
(0044]
In the culture substrate prepared in this manner, ~
plurality of the holes 101 are formed on the culture sheet
15 100 formed at the bottom of the frame bOdY 111, and a
plurality of projections constitute.d at the. bottom l04 of
the hole include a plurality of microprojections 103
(hereinafter also referred to as projections, pillars or
nanopillars). Moreover, the diameter of this hole 101 is
20 used as a hole diameter 105. In the culture sheet 100, the
hole 101 including the above-mentioned pa~tition wall 102
and the plurality of proj ections. 103 formed inside the hole
101 are formed from the same material integrally. It
should be noted that the shape of this hole 101 is not
25 limited to round, but may have other shapes such as a
~Sli~~~ ~QeB~~
..:. .,:;' . ,',' ,. '<" .' ',,-
"'
~gg~~J
~~ thi~ m9ij~~~! tB~ Qg~~ bq~ ~n~¢Uq~~~ tb~ ~~ft~t~Rn
w~l~ 102 anq the plu~ality ~f projections 103 formed inside
5 the hole 101 are formed integrally from a single material
which has no adverse effects on cells as a culture sheet,
whereby cells can be grown with no foreign substances
adhering to cells in the cultu~e step. Furthermere, since
cells are grown in each of the partitions, cells of a
10 homogeneous size ca~ be formed.
(0046]
within the partition arranged in a surrounding manner, and
therefore cell movement, which is the ability inherent to
15 the cells, is promoted, and cells are grown by the movement
is possible with no influence of disturbance (stress) by
rotation culture or th~ like.
[0047]
20 When a culture region is to be formed while these
holes 101 and projection assembly 103 are provided
separately, they need te be joined by adhesion or welding.
For example, when joined by adhesion, adhesive components
enter into the culture region, which may adversely affect
25 generated cells.
%9~~8Y~f~ wh~n w~+g~PT ~~ ~9 p.~ ~eF~Q~~q( thy i~ner
q.1,"qmete.r of the hole 101 is a hyperfine regiqn cii,.a:ql,eter on
f1.
th~ ce~+ format.ion lev.el, and therefore it is verN
5 difficult to perform welding while forming a target cell
region and not damaging the partitions and' projections.
When the partitions and projections have damages and
defo~ations, unwanted stress may be applied on the cells
in the process of cell formation, and the movement of the
10 cells themselves may be impaired.
[0049]
'rherEr~ore, the bo,1.e l0I. Which. fom~ the c\llture
region and the projections 103 are preferably fon!ed
integrally by forming integrally in such a manner, it is
15 preferable because cult\lre exclUding the influenteof
,
unwanted components other than those required for cell
culture can be performeci.
20 constitution diagram and a principal part cross~sectional
view of a multiwell plate with the culture sheet of this
E~ample are shown.
[0051]
r.ig. 14A ~how~ ~p ~ppearance per$peqtive view and ~
25 bottom view of the culture substrate in this ~~§mple.
24
Fig. 14B shows a top view and upper and lower side views of
the culture substrate. Herein, illustration of lert and
,., .
right side elevational views is omitted since it~ form is
obvious from the appearance perspective view.
5 Fig. 14C is a partially enlarged view and a partial crosssectional
view, which show an A-A, a B-B partially enlarged
view, a C-C, D-D partially enlarged view, and an H-H crosssectional
view.
Fig. 14D is a partially enlarged view, and an end view,
10 ,which show an E-E, F-F partially enlarged view, and a line
G-G end view.
[0052]
The article shown in Figs. 14A, 14B, 14C, 14D is a
culture device (culture container) for cUlturing cells of
15 humans, animals, plants and others, and is constituted by
the culture sheet 100 and a retaining member (frame body)
111 which retains the culture sheet 100.
[0053]
A plurality of the holes 101 are formed on the
20 surface of the culture, sheet 100, and is provided at the
bottom of the inside of a cylindrical hole portion 111a
formed in the retaining member.
Furthermore, culture regions having a plurality of minute
projection portion 103 within the partition portion are
25 formed respectively. When target cells to be cultured are
25
It ~g9~9 ~9 th~ ~p~19~ 9t ~h~ ~91~ ~qrt;9.~ **+~! ~, ~ge!q ~9
tb~ ~h~~~ ~1.f:r:~e<::~ ~9tm~~9' ~h~ ~JJ.ltH;t.7~ ~~~~8!}~ w;~fii.ri'~b~
h8+~ *Q+~ tQ~ t~;~~~ G~+l i~ ~~~~;n~q ;p ~he ~+~r~+~~y Q+
!5 [OO~4]
Mo~eov~r, the culture suPstrate of this ex~rop~~ $~qWs'
.: I. ' , ... ,\ )"': ,:',:',.'" -~' '1, - i: '::-.t:: ,~: 'i},,'.\,~',:~:~'1' ,-,,' ",- -'. ':~
an example in which the culture sheet is welded from the
back side of the frame body 111, and the frame body 111
which is a retaining me~er and the culture $heet 100 are
10 welded via a joint ll1~~
portiGn 11la, and the culture region is not affected bY the
welding.
15 ~~a~p'le" th.~ joining method is not limited to tais, and
oth~~ joining methods cap be also employed since joining
does not affect the culture region itself with other
~Q ' ;~ ~9g;t~qp, ~~ ~~~ ~~Q~~+~~~ gf th~~ ~~~~l~, t~~
~+~~~ R99¥ +11 h~~ t~~ f9~~ of a §~9+~" ~~g ~t +~~§;9.f
tb~ *9~F ~~~~~~ ;~ g~t 9~f~ ~n~ f9~~H~qp 9+ t~~s qu.t
sU~f~~e ~1~3 f~9il~tate$ ~Pecificati9P of the position of
~~~. ho+e p.9rt~o~ q~ the sYQ~trate py th~ g~~rat9r who
25 ~~~f9f~~ yu~ture.
rb!~ q4t f~g~ ~~ oQ,t ,,~~~~t~~l, ~n~ 9# ~84~~~ m~y. ~~ 9; m~y
~ . '. _ ".. ., ': . - . ~ . - " ' . : 'i. ;..... . . ,
~B*~~r~ ~~P~~;~~7{ ~~~g~ 9~P ~F~Y~~t ~h~ ~p~~~t9F ;7~~
~~~~P~9~~q+y SA~~~ng ~~g g;opP~p~ t~~ §Y9~tf~t~ ~~Q PF~y~pt
5 Qtb~r ~G9~4~nts d~r;ng th~ ope+~t~qp.
[OPp6J
.*~~g+~ ~ ~hQ~~ ~p ~~~p+~ gf ~~~t;~~~!g~ Qt ~~}t~ t9
10 tiss~e GuLture using the cu+ture substrat~s pro4uced in
Gells subst~tut~ng a.nimal ~~periments. In addition, when
15 the thus-formed 3D tissues are subjected to various tests
it is necessary to verify in advance whether the 3D tissues
retain activities to withstand the tests. In this case, if
the formed spheroids are held in the predetermined position
20 with high reproducibility, it is expected that they are'
suitable for high through-put screening and various tests.
(OO,?7]
cel+s (iPS cells)a.nd embryonic stem cells (ES cells) to
25 cause th'm to differentiate into target cells, 3D tissues
~~m~!!;:~9~!~~ ~p·t~~~~~,~,;:q~§ p~~n- 9,~p\\~;}g~g ~~~P !P' tb" ~~!*q
.. ," >,' .",' . c- _ ,••. ;;-: -, ;'. - .,' .' ,- "
~~~mBt~ 9~ f9-~~tq~ ~p. ~i§§Hi~ ~~~p.~ ~h~ ~9~~~ s.li4~ ~~
$ ~e~t19~+~~ ~~ §P9~ h~F~+PI PHt ~n~ ~~~~~tt~+ ~~f; q~ g~+l
g~J.t1J.:!i'e ~$ U91:: es.peg;ally ciiffe.t:ent even fqr i;l ltt1J.l1;:iwel,J..
plate. In this excunple, an eXcunple using rat hePatocytes
is shown, but as mentioned above, it is ~pplicable to cell
strains of various animals and plants, and cell strains are
10 not especially limited.
[QQS8)
in situ collagenase perfusion technique. The detail is as
follows: The q~domen of a Fis~er 344 male rat (7 to 10
15 weeks old) is oPened under pentobarbital anesthesia, and a
c.atbet.er is inserted into the portal vein to inject a pre~
PeJ::'fusate (Hanks' solution not inq1.Ud,ing Ca2+ or Mg2+ and.
2Q
right atrium is incised, and the pqstq~va+ vein in the
19~er liver is clipped with a clcunp to perform perfusion.
25 Perfusion is stopped after it is confi rmed that the blood
~e oval frqm. the liver has be~n fully cq~ducteq. ~pe
-co" ~'" .., -,," '" ", . """ '.•~. ,"', • " ':. ",', .' ";".' ..:.'.".\. "'; .' : "" "- . '-,_ .':" " - . . .. ' ,": ">., .;, ".,-., .. '. " ."-- '.•" -." " ..... ' .. --.~.
p~ tM~~t~ i.~ ~~~A~n~~g to a Gpll,agenase §o+U~~9P ~p per~9P~
pe tus~on.
5 ~~rfu§i9n is p'er~orme~ usi~g the ija.nk$~ sO~Htion.
containing 0.05% of cOllagenase in this ex~ple, but this
solution is not necessarilY used. perfusion is stopped
after it is confirmed that intercellular tissues have been
digested by collagenase. The liver is separated, cut into
10 small pieces in a cooled Hanks' solution, and is dispersed
into cells by pipetting~ Subsequently, undigested tissues
are re~oved by gau~e filtration. The ce+1 suspension is
repeatedly centrifyg~d ~t $0 ~ for ~ ~inut~ several times
to remove nopparenchymal cells. Subsequen~ly, damaged
15 nepatocytes are reffiOveq py GeP~+i.tu9~1 $~pav.at.~OQ ~t 500 G
fqf ~ minutes ~sing an isotonic Fercoll solution. The
survival rate of the obtained hepatocytes is measured by
the trypan blue exclusion method, and the hepatocytes with
a survival rate of 85% Or higher are used for culture.
20 ijerein, the hepatocytes with a survival r~te of 8~% or
higher are used fOr cUlture, put it goes without saying
that this condition is not necessarily used. Preparation
of the hepatocytes is not necessarily limited to the in
situ collagenase perfusiOn technique.
25 [0061]
n~p~t;89¥~~~ ~~ ~bQW1t i~i W~9-' ~~?,
j.'
~'.
+D t.~~ f+9~9B~ft 9~ fig. 6, ~;~~~I, t~~ *q8t~f~~A
5 *+~ i§ ~~p.~i~q to tb~ 9~+t~P~ ~q~~t qt th~ qtqmb~; $Lide
type produced in Example 1. ~ 1 to 1~5~ml portion of a
diluted solution which has been produced by diluting type I
collagen dissolved in a weakly acidic solution with sterile
water to a predetermined concentration is added to the
10 chamber slide mentione¢ ~bove (Fig. 6(a)) ~ ~e~t, a
¢eQQmBre§sion oper~tion is performed in order to cause the
added type I coLlagen to be ad$orbed onto the nanopillar
sheet 100 completely (Fig. 6(b)). The decompression
operation is performed at 0.04 atmosphere or lower using a
The decompre$$ion ~~me i$ not particularly limited, but the
d~90mpre$~ion is Performed for ~O minutes i~ tAi~ ~~~mp+e.
*b~ CQP,§t;t~ti9n of ~he ~pp.~;at~$ U.$ed fo~ geCo~pressi.q~ is
not particplarly l~~ited~ ~erein, the r4nge of the
20 predetermined concentration of the diluted solution is 100
(ng/ml) or higher and 10 (pg/ml) Qr lower~ The
this range is suitable for spherical 3D tis$ues to form.
Finally, an excess of type I collagen is removed, and PBS(-
25 ) 119 is added thereto (Fig. 6(c)). This operation is
3Q
tt E~;f9~~~ t~+~@ ~~~~~~ ~Bg ~~ ~~G~~~ o~ ty.e~ I qR+~~~~~ i~
~~~b~g~
~~~~~~9¥~~~ +~Q ~t~P,~F~~ ~¥ t~~ ~n §~~~ 9~+*~~~n~~~
~~~+H~~8~ ~~9g~~q~~'~~ ~pq~~~~~~~~9P,~9 4f~ ~~~e~ng~g ;~ ~
-:;" '.. .. ,,' . ':' . ." .' -.- ,... . ',",' :.,. -', ,-'. ", ,,".'.' -,-, ", .', "'.,": -. '< •
W~g!wm ~g+f ~p¢ t~~ ~~~~~n~i9~ i~ inQ~y~~t~4 QP ~h~ Ni
sb~~t w;th WYP~ I qQ~+~g~n prepa+~q ~s st~t~4 aQOv~ ~pp~ied
th~~et9 s~mi+ar+y (Fig~ 6(q)) ~ The ~eqi~ is ~ot
~~~t~g~1~~+¥ *!mit,~4, p~~~ ~ Wi+*~~m~ ~ m~Q~~ ipql~Q;ng a
10 me4ium containi~g serum (~C.~), insulin, and de~amethasone
(hereinafter r~fer~ed to as medium (~ncluding 10% FCS)) is
~Seq. In this E~ample, a Williams E medi~ containing 10%
Fes, 8.6 nM insulin, and 255 nM de~amethasone is
particularly used. After inoculation, Gulture is started
15 using a C02 incubato~ under the conditions of 5% C02 and
~7 qc, the first m~dium exchange is perfa~ed after 18
hours Or mo~e has ~l~psed, and medium eXCAange is performed
every 24 hours penc~forth. ALthougn the medium used for the
f;G$ remG.ved from a medium (containing 10% FC$) is used.
(0064)
M9~eover, th~ inoc~+atiop de,n~1ty 0t A~Pa,t9Cyt~~ W~~
~~ ~~t tp +~t05 Q~~l~/m~ ~~ thi~ ~~a,mple, QMt i§ ~9.t +;~1t~q
It ~9 th~~ 8P~9rP~+~t~g~. ~~f~~r, thr 9H~;~F~ §p~~t +P9 ~~~q
~9f gH+tH;-~ ha~ ~ ~++l~~ h~~~ht.~ g~+J,~t' gA~m~~~~' ~B~ i?~t;L~F
p.~~e~ q; +~o ~, 2·9 ~r ~~g 4~q ~~ ~~~~~~t~R~+¥1 P4t t~e
Y~t~~S ~r~ n9t lim~~~g to th~~~.
S [Q985]
~or~qy~r, 4h~ CQr~~~tF~t~9B of I~P~ ~ cq+t~g~p ag9~q
to the cu+tur~ she.et i~ set to 100 (ng/ml) i~ this ~x9mple,
b~t may be a concentration other tha~ this. $PQ~roids may
be formed at a concentration other than this concentration
10 depending on the conditions of the cells. The cells are
cultu~es for 96 hou~s in total, whereby 30 tissues 122 are
;Q+.m~g (f,ig~ p(~)).
fQQ6t3)
Fig. 7 shQW~ ~ phptqgraph Q; th~ #~§~+t~ 0+ act~al
15 culture of hepatocytes using the above-mentioned culture
sh~et having ~ hole diameter of 200 gm. ~s Can be seen from
Fig. 7, spherical 3D tissues 71 haviRg such similar sizes
are formed in the hOle~ 70 with no special chemical applied
onto the surface of the culture sheet a~d by stationary
20 G~lture h~vi.ng li~t+e ~treSS on cells~ ~his culture method
supposedly does not deteriQrate the activity of the cellS
or~ginallY re~atned, a~q A~ therefore effective for cell
a~~ays and the like.
25 Example 4
f+g~ a ~how~ ~ V~Fi~~t 8~ ~x~mpl~ 8~ th,~ 9Y~t~~~
-: :-
5 arrang~~~nt patteru o~ ~~9jeQtion~ whi.ch provi.des
di~ferences in the migration and adne~ion of cells in two
stages, as in Fig. 8(a}, in a ~anner of s~rrounding a first
arrangement pattern 125a with a second arrangement pattern
12~p, 3D tissues or 2D tissues are tormed on the first
10 arrangement pat t ern 12~a (for example, pear the center of
the ho'Le) •
(0068]
Contrarily, an example is shown in which, as in Fig.
,8(b), by arranging in two stages in a manner of surrounding
15 the second arrangement pattern 1250 with the first
arrangement pattern 125a, 3D tiss~es or 2D tissues are
formed on the second arrangement pattern 125b (for example,
the periphery of the hole). It should be noted that 124
[0069]
in the central portion of the hole 124, but also arranging
the same as in the culture sheet 126 of Fig. 8(c), by
25 surrounding, for example, 4 portions of the first
It ~~t~B9~m~~t ~~tt~r~§ t?7p w;t.r ~b~ ~~9Rn~ ~~~~v~~m~~~
B~tt~fB ~?7~, ti$~H~~ hav~~g §~m~l~~ si~~~ ~~p ~~ fQ~~~~ On
t~~ f;f§t ~rrapg~m~nt 2~ttern. I~ th~~ ~a.p~~~, the
5 arrangement pattern can be an optimum pattern of
arrangement depending on the purpose to perform culture.
Similarly, Fig. i8(d) shows a culture sheet 128 in which the I
arrangement pattern is set to be multi~stage patterns 129c,
10 (OQ70J
patterns (hereinafter referred to as pillar patterns) of
qescribed. As shown in Fig. 9, 11 types of arrangement
15 patterns have beep shOWn as examples. As can be seen from
the same figure, the~e are 11 types of arrangement patterns
tq ?O.Q ~ ~nd +rq~ O.~6 to 40.0 ~~ respectivel¥, but the
pillar diameter anq pillar pitch are not limited to these.
fO [007~]
[0072]
It shoulq be note,d that i~ tAe culture on ~ flat
25 plane with no pillar pattern, many cells are discharged
I
~+g~~ ~~t~ t~~ ~~gi.~ w~~~ tn~ m~9~~ t~ 9h~~~~g q~f;~~ th~
i - . ' '~."~' " .'',\:' :.., -.' ',- .'" '."', ': -.. - . ,:.~\i.: - '.: ,"' ., ' . , : .: :.'. - - . .,'., . .'... ; ,
g~+tg~~'. ~rd ~p~~~f9;~ R~~~;~q 8~tt8~~Q 9~~~~ 9.~n~9t ~~
~~t~~t~~~~y. qRt~~P~g,~ A9g,~+9~ng~~( q9 !~~g§~;~~;qQ ~§
". " - -'. .' . c· ',' • ': :-', _.' " -., ..'_ ,_,'0' :. '. "'".' . ,- ',"; . _,. • ..~ ...
[0973J . ~' . - .:-.~
rig. lOA are tig~r~~ which shoW 1:pe states of the
cells wh~~ c~ltpre is performed using the c~lture sheet 100
wi th the double pit ch relative to the pillar diameter. 1\s
a result, when the pillar diameter is 0.18 pm, 0.5 pm, and
10 1.0 pm, flat tissues which are not spherical are adhered at
the bottom of the substrate, while, when it is 2.0 ~ and
5.0 ~, 3D tissues which are spherical are formed on the
suPstrate.
[0074J
15 Oomparing the spherical cells for-med in the substrate
with the pillar diameter of 2.0 ~ and 5.0 pm, the
substrate with the pillar diameter of 2.0 pm had more cells
aQhered onto the substrate, indicating that it is in a
staple state~ That ;5, it can be seen that as for the cell
20 adhesion, the greater the pillar diameter, the lower the
adhesion and the more promoted the movement by cells.
{0075J
Fig. lOB is a graph which s.hows, as for the number of
3D tissues (spheroids) of the hepatocytes formed on the
25 sheets with each of the pillar diameters, the results
It q+ou~~g BY di~~~er 9.f. th~ ~ph~~oig~ f9~m~~· Tp~ ~~e~ of '" . .,':: ~. ',:.' '. -.:. " .. , ',,:~, ". . ,
[OQ7p]
I~ t~~ 30 tis~uee of h~p~to9¥~~~, in C~~+ ~ep~ye
5 ipt~nqed for dr~gs ecr~~ping, an.q t~e to~i.qity. ~nd
ID~tabolis~ t~ets whiqh pan subetitute ~pimal exp~riIDents in
the innovative drug ?,ey~lopment field, cells having
4~proe~~rs of ~O to 10Q microns are preferable. Ip this
ex~p~e, it can be seen that the numPer of the formed cells
10 of this size is the most in the case of t~e substrate with
a pil+ar di~eter of 2~0 pm, indicating that this pillar
di~eter is preferable.
[9°77]
15 w.~s stated that the case where the pillar diameter is 2.0
50 to 100 microns, but the pillar diameter is not limited
e~~ination, it was foun.d that a greater number of cells
20 with stable shapes are formed compared to the flat state
with nq pillar fo~eg. Thus, the fQ~ or a4hesiop to the
sllPstrate of Ce+ls or tissues ~Qrmed fro~ cells can be
freely changed by the difference in pillar pattern.
[Q078J
25
5
~~~p+~ 9! Fi~. 8, Q¥ ~~~ergin~ in t~9 ~t~ge~ ;~ ~ ~a~ner - ;,
ot s.~rro~~d~n.9 the f~rst ~r~an.geme~t p~tt~~n ~ith q s.ma+ l
pil~ar Qi~me,~e~ (pi~lar pitGb) py the ~eGQn.g a~rangement
pattern with a large pillar diamete~ (pi+lar pitch), or
ar~anging in multiple stages, tissues having target shapes
can be formed in target positions within the holes
utili~ing cell adhesion and the motion c.baracteristics of
[007~]
10 Moreover, by decreasing the heights of the
n~mopillars having the Same size of the piJ,.lar d,iarneter
from the periphery tow~rd the ~entral po~tion of the hole,
it is possible to prqvide a differenee in hei~ht gradually
in a manner of in9li~irg, to promote the movement of cells
15 $0 that they gathe~ in. the central portion by gravity and
form tissues.
[0080]
~ig. 11(a) s~o.~s a cu+tur~ s~e~t l~O ~h~ch ~s a
variant in Which a difference is provideQ in the h~ights of
20 the nanopi;Llars gradually. At this time, unlike in a
normal U-shaped culture container,. there is produced an
effect that the cells are retained in the cente~ by the
presence of the pillars. In addition, as in the culture
sheet 131 of Fig. 11(b), it is also possible to promote the
25 effect stated above by providing a difference in pillar
It q4~!F~; ~y~p ~p ~h~ ;p~+~n~~;8n·
(_Og~~l
~~ th~ v.~~;~qt pi ~iq. l~, tb~ h~;ght ;~ qha,~g~g
• • . - " .- " .-"" ~ • '. " :i' ',' _.. ' : '. ' .' , _ . '.. \. ',".,. • • :.', '-.' . .
g+~QU~+ly to ~moQtb~p tg~ incl;n.~tiqn, put a co~stitution
5 in whicp the height is sequent~allY cha,n.ged stepwise may be
ca:Lso etnploY~d.
[09~?]
~preqy~r~ ~ pl.'ur~lity o~ h,o~es g~the~ to tQ+~ ~ ,- - " -, ." . ". . .." .. - . - , .}.;
cUlture surface (square snape in the case of the chamber
10 slide, round shape in the case of and the plate), but in
the cultu~e, a difference occurs in how 3P tissues ar~
formed in the central portion and peripheral portion of the
cUlture surface by the influence of the surface tension.
Tbat is, altnou9h 3D tissues are formed in the central
15 pqrtion of the cUlture surface, 3D tissues may not pe
formed in some events for the reason that the amount of the '" _. • • -'. -, ,. ' -' ", . # ' '. . .' ••.. . ~. - . _.. •..• ,., - ;-: ....'
m~diuw is iP9~eased pf the portion by the surface tension
in the peripheral port;on, the amount of oxygen supplied is
lowered, or the high water pressure is app~ied~ In order
20 to avoid this phenomenon, the cUlture sheets 132, 133
retaining the hole ~tructure may be proquced only in the
cen~ral portion of the culture surface as shown in Figs. 12
(a), (b).
[0083]
25 ~y f~r~iBg th~ c~ltu~e sheet in thi~ manner, the
tt G4~~~,~ ~HP~t~~t~ h~yip~ pi~h 9~~t4~~ ~tf;qi~ppy qPQ ~~~tl~
P~o9u~tio~ load can be ~ch,ieyeq.
5 (0084)
f.~g~ Iph, F~~. 15,~~ [~g. ~?S~ ~Qq f.~~7 t?p ~g0o/ th~
culture sheet of Ex~mple 5. In Example 5, among the
various variants shown in Example 4 of fig. 8, an example
is shown in which the first ~rrangement pattern 125a is a
10 flat structure, and a culture sheet having a pattern in
which projections are ~rranged in the central portion of
the hole is applied to the, chamber slide which is a culture
15 culture regions consist of the first region and the second
region surrounding the same, projections are ar~anged on in
the fir~t region, and projections are not fOrmed in th,e
~econd region, spheroids which are 3D tissues having
20 the cult~re region corresponding to ~he tirst region,
whereby the spheroids c~n be retained in the target
position.
(0085]
• Herein, an example in which projections are arranged
25 near the center in the culture region is shown, but the
e 9~m~~2,:' n~~~ pot l;l~ n~9«ess~ril¥ il1~+l.;q~~, ~B-c:i ~; ~9~$,
w~t~o~t saying that p~ojeqtion$ ~~y be ~rFan~~d ip ~
d~si+eq r~9iQn in th~ culture regiqn~ Mo+eover, ~ltho~9A
~n e~~~p~e in wh~qp the p~ojectiqn regiop of an approximate
5 rhombus shape and circle shape is formed is shown, it goes
without saying that the projection region mgY be in the
shape of a square or a po~ygon.
[0086]
holes 151 constituted by a plurality of partition
structures 152 in a single culture sheet. The
config~ration that the inside of the hole 10+ cQnstit~tes a
culture region by a cell tissue formation unit partitioned
15 by a pa+tition wall is the same as in the above~mentioned
Example. A pluralit.y of projections 153 retained at the
bottom 154 of the hol.e 151 includes a pl.~rality of
microprojections. Moreover, the diameter of this hole 151
is set tube a hole qi~~~ter 155. Preferabl.Y, in the
20 9~~t~+e s.heet 159" ~he AQ~~ ~51 inc~u4iP9 the aPQvemention~
d partition wall. 152 and a plurality of p+ojections
153 formed within the hole 151 are formed from the same
material integrally. It should be noted that the shape of
this hole 151 is not limited to round, but may be another
25 shape such as a square, as in the above-mentioned Example.
40
In ~ suit~ble ~~p~et qf ;b;~ ~x~p+~, ~~ ~A9wn ;~ the
cu~tur~ ~~~et 150a·Q~F~g. 1~~, cu~~u;@ Ib"t~ b~x~Oq,
p~~~~~ h~ight, pil+~f di~me~~r, ~p4 ~i++~f pitGh of *.0 ~,
5 1.0 gm, 2~O ~ a~q, l~O gm, 2.0 pm, 4.0 ~, respectively,
anq a di~eter 9f th~ as~~~+¥ 9 f p~ojeqt~Q~ po.rtion~ ot
200 ~ (panopi+l~~s o.~ th~ entire ~u~f~ce), 150 ~, 120 ~,
1.00 ~, ~O ~, 69 ~, 40 ~, ~Q ~ c~p be USeP.
(OQS8]
10 ~s ,~own in ~n e~+~~ged port1o.n 1~0~ o.~ r~~~ 151,
providing a qul.tu~e ~pb$t~ate in whic~ the torm~t~on region
(constitutional proportion) of projections by in the hole,
that is, a cell tissue formation unit partitioned by a
15 a test substrate for grasping an optim~ formation rate of
projection regions in Example 7 described later. An
optimum constitutional prqportion ~ay vary depending on the
cell strains and desired si~e intended for culture, and
20 culture substrate is useful since it affects the culture
It should be noted that the hole ",,,'" ....-';'''\.". , ..'.-:; ,- ,.- ' .... "."-, ',,', "'.-' ..,..... ..... ,-, .... .... ",
151a indicates a hole ~n ~hich no projection is fo.~med.
(0089]
The culture sheet 150a is formed from a material
25 ~piph does not adversely affect cells, anq it is, in this
20
It ~~~~pl~, ~91y~ty~~~~. ~Q~§y.~r, ;t ~q~s ~;~po~t ~~¥~n~ th~t
~b~ mqter~al is not limit~g to P91ystyrene.
(0090)
As a typic~l example, a S~M im~ge ~n. wbich the
5 diameter of the assembly of projection portions is 80 pm is
sl10wn in Fig. 15C, ar).(j ~n SEM image in which the di~eter
is 20 pm is shown i~ Fig. 15D. In eacp of Fig~ 15G, and
Fj.g~ 15D, 156 a:n.d 158 repres~nt ~ hole, While 157 aIld 159
represent a projection ~ssembly.
~p [Qo.~ll
In th;s m.~:n.P.~r( th~ hol~ 15! i~~lu4~ng th~ P~rtit~9n
wall 152 and the pluFality of projectio~s 153 formed inside
the hole 151 are fOrmed integrally from a si~gle material
Which has no adverse effects on cells as culture sheets 150,
15 150~, whereby cells ca.~ be gro~ wito no for~ign substances
aqhering to cells in th~ CUlture step.
Furtber~ore, since c~lls. ~re gro~n in each of th~
B~rt~t~9~~' ~ell~ .o~ a bqmogeneo~s si~e qap. be fq;~eq.
[0092)
Moreover, a plurality of Projections are prqv~deq
within the p~rtition wall 152 arranged ~n a surrounding
~~~~~r. Therefor~, cell ~ovem~:n.t, Wh~qb is tl1e ability
i~berent to the cells, is promoted, and cells are grown by
tbe movement so that a cell culture which can maintain the
25 cell activity is possible with no influence of distur~a~ce
42
It (stress) by rotation culture or the like.
[0093]
When a culture region is to be formed while these
holes 151 and projection assembly 153 are provided
5 separately, they need to be joined by adhesion or welding.
For example, when these are joined by adhesion, adhesive
components enter into the culture region, which may
adversely affect generated -cells.
In joining by welding, the inner diameter of the hole 151
10 is a hyperfine region diameter on the cell formation level,
and therefore it is very difficult to perform welding while
forming a target cell region and not damaging the
partitions and projections. When the partitions and
Frojections have damages and deformations, unwanted stress
15 may be applied on the cells in the process of cell
formation, and the movement of the cells themselves may be
impaired.
[0094]
Therefore, also in this Example, as stated above, the
20 hole bottom 154, partition wall 152 and projections 153
constituting the holes 151 which form the culture region
are preferably formed integrally by forming integrally in
such a manner, it is preferable because culture excluding
the influence of unwanted components other than those
25 required for cell culture can be performed.
43
e [0095J
In this Example, a culture sheet in which the pillar
diameter, pillar pitch and pillar height are 1.0 pm or 2.0
pm, 2.0 pm or 4.0 Pm, 1.0 Pm, respectively,. was used, but
5 as will be described later, the culture sheet may be one
with other specifications than these. The height of the
partition structure is 70 pm in this Example, but this
value is not necessarily used, and suitably the height may
be such that the formed cells do not get over the partition.
10 [0096]
The culture sheets 150, 150a in this Example are
..
produced by a method similar to that in Example 1, and
therefore detailed description of the production method
will be omitted herein. In addition, also in this Example,
15 the chamber slide 109 with the culture sheet 150 affixed as
shown in Fig. 3 can be produced, and it goes without saying
that a chamber slide having an overall constitution and a
principal part cross section similar to those in Fig. 13A,
Fig. 13B, Fig. 13C can be obtained, and therefore
20 explanation will be omitted herein.
Example 6
[0097]
Subsequently, Example 6 will be described with
25 reference to Figs. 4 and 5. This Example shows the
5
44
constitution of a multiwell plate with a culture sheet
using the culture sheets 150, 150a described in Example 5,
and a production example thereof. The constitution of the
multiwell plate and a production example of the same have
been described in Figs. 4 and 5, but this Example is
basically similar to Example 2 except that the culture
sheets 150, 150a are used in place of the culture sheet 100
used in Example 2.
[0098]
10 Fig. 4(a) is a bottom view of the frame body 111
constituting the multiwell plate. The frame body 111 which
is a culture sheet retaining member is such that has 24
cylindrical hole portions lIla in total, arranged in 4 rows
and 6 columns, are formed in an area measuring about 125 rom
15 in width, about 80 rom in length, and about 20 rom in height.
The material used is polystyrene.
[0099]
The number of holes formed on the frame body normally
ranges from 6 to 1536, varied depending on the use, and
20 therefore the number of holes on this frame body is not
limited to 24. The material of the frame body is not
limited to polystyrene either.
[0100]
In producing the culture substrate, the frame body
25 111 and the culture sheets 150, 150a in Figs. 15A and 15B
45
are joined by ultrasonic welding. The procesS and
constitution mentioned above are the same as those in
Example 2, and their explanation will be therefore omitted
herein.
5 [0101]
In the culture substrate prepared in this manner, a
plurality of holes 151 are formed on the culture sheets 150,
150a used in place of the culture sheet 100 formed at the
bottom of the frame body 111, and a plurality of
10 projections constituted at the bottom 154 of the hole are
constituted by a plurality of microprojections 153. In the
culture sheets 150, 150a, the holes 151 including the
above-mentioned partition wall 152 and the plurality of
projections 153 formed within the holes 151 are formed from
15 the same material integrally. It should be noted that the
shape of this hole 151 is not limited to round, and may
have other shapes such as a square shape.
[0102]
In this manner, the hole 151 including the partition
20 wall 152 and the plurality of projections 153 formed inside
the hole 151 are formed integrally from a single material
which has no adverse effects on cells as a culture sheet,
whereby cells can be grown with no foreign substances
adhering to cells in the culture step.
25 Furthermore, since cells are grown in each of the
.J
46
tt partitions, cells of a homogeneous size can be formed.
Moreover, a plurality of projections are provided
within the partition arranged in a surrounding manner.
Therefore, cell movement, which is the ability inherent to
5 the cells, is promoted, and cells are grown by the movement
so that a cell culture which can maintain the cell activity
is possible with no influence of disturbance (stress) by
rotation culture or the like.
[0103J
10 As stated above, also in this Example, the holes 151
which form the culture region and the projections 153 are
preferably formed integrally. By forming integrally in
such a manner, culture excluding the influence of unwanted
components other than those required for cell culture can
15 be favorably performed.
[0104J
The overall constitution diagram and principal part
cross-sectional view of the multiwell plate with the
culture sheet of this Example are also as shown in Figs.
20 14A, 14B, 14C, 14D as in Example 2, and explanation will be
therefore omitted herein.
Example 7
[0105]
25 Subsequently, Example 7 shows an example of
47
application of cells to tissue culture using the culture
substrate produced in Examples 5 and 6. An example of
application of cells to tissue culture using the culture
substrates produced in Examples 1 and 2 was shown
5 previously as Example 3 using Figs. 6 and 7. The
difference between this Example and Example 3 is that a
culture substrate in which the culture sheets 150, 150a are
used in place of the culture sheet 100 is used. Since
explanation is common for other point,. explanation will be
10 therefore omitted herein.
[0106J
It should be noted that in this Example, the
inoculation density of hepatocytes is set to 5 x 10 5
cells/ml, but is not limited to this concentration. Herein,
15 the culture sheets 150, 150a used for culture as previously
explained, have a pillar height, pillar diameter and pillar
pitch of 1.0 ~, 1.0 ~, 2.0 ~ and, 1.0 ~, 2.0 ~, 4.0 ~,
respectively, but the values are not limited to these.
(0107]
20 Moreover, the concentration of Type I collagen added
to the culture sheets 150, 150a was set to Example 100
(ng/ml) in this Example, but may be a concentration other
than this. Spheroids may be formed at a concentration
other than this concentration depending on the conditions
25 of the cells. In this Example, as shown in Fig. 15B,
48
tt culture sheets having a pillar height, pillar diameter and
pillar pitch of 1.0 ~, 1.0 ~, 2.0 pm and, 1.0 ~, 2.0 ~,
4.0 ~, respectively, a hole diameter of 200 ~m, a diameter
of the assembly of projection portions of 200 ~
5 (nanopillars on the entire surface), 150 ~, 120 pm, 100 pm,
80 pm, 60 ~m, 40 pm, 20 pm, respectively was used.
Needless to say, the hole diameter and the diameter of the
assembly of projection portions are not limited to these
values.
10 [0108]
Figs. -16A, 16B, 16C, 16D show the results of the cell
culture for 96 hours in total using the culture sheets
having these patterns. That is, the graphs of the results
of measuring the distance between the center of the hole
15 and the center of the spheroid and verifying- the hole
center retention rate of the spheroids are shown. Figs.
16A, 16B, l6C, l6D, as illustrated, correspond to a square
20
arrangement with a pillar diameter of 2.0 um, a triangle
arrangement with a pillar diameter of 2.0 um, a square
arrangement with a pillar diameter of 1.0 um, and a
triangle arrangement with a pillar diameter of 1 . 0 um;
respectively.
[0109J
The distance between the centers of the hole center
25 and spheroid is indicated in 3 steps of 0 to 19 pm, 20 to
49
39 pm, 40 ~ or more on the horizontal axis of each graph,
and the proportion of the number of spheroids occupying
each range in the total number of spheroids is indicated on
the vertical axis. As a result, in all of the patterns
5 examined at this timet the sheets having a diameter of the
assembly of projection portions of 100 pm or 80 ~ had
higher rates that spheroids are retained closer to the
center.
[0110]
10 It has been shown that an optimum rate of the
diameter of the assembly of projection portions relative to
the hole diameter is 40% to 50%, but it is not limited to
this value depending on the cell strain and culture
conditions. According to the experiment results, when the
15 rate is from 20% to 75%, more than half of the spheroids
also fell within the range of the distance between the hole
center and the center of spheroid from 20 to 39 pm. The
rate may be therefore within this range from 20% to 75%.
[OlllJ
20 Figs. 17 and 18 show the results of the culture
sheets in which a pillar height, pillar diameter, and a
pillar pitch of 1.0 pm, 2.0 pm, 4.0 ~, respectively, and a
diameter of the assembly of projection portions of 80 pm
and 20 pm, respectively, as typical examples of phase-
25 contrast micrographs of the results of culture of the
50
tt culture sheet of this Example. The numbers 170, 180 in the
holes 156, 158 in the figures represent spheroids. As
shown in Fig. 17, when the diameter of the assembly of
projection portions is 80 ~, the spheroids 170 having
5 almost the same diameter were retained in the central
portions of the holes 156. In contrast, as shown in Fig.
18, in the sheet having a diameter of the assembly of
projection portions bf 20 ~ the spheroids 180 were not
retained in the central portions.
10 [0112]
AS can be clearly seen from the results described
above, it was found that according to the culture substrate
and culture sheet of the present invention, spherical 3D
tissues having such similar sizes are formed without
15 applying any special chemical on the surface of the culture
sheet and by stationary culture which causes little stress
on cells, and spheroids having similar sizes are retained
in the central portions of the holes by appropriately
setting the hole diameter and the diameter of the assembly
20 of projection portions.
Reference Signs List
[0113]
100, 123, 126, 128, 130, 131, 132, 133, 150, 150a...Culture
25 sheet
51
e 101, 124, 151, 156, 158...Hole
102, 152_Partition wall
103, 153, 157, 159...Proj ection / projection assembly
104, 154...Hole bottom
5 105, 155...Hole diameter
106...Pillar diameter
107_Pillar pitch
108_pillar height
109 Chamber slide
10 110 Surgical glue
IIL.Frame body
1I1a...Hole portion formed on frame body
112_Projection for fixing film
113 Rib structure
15 114 Culture sheet hole
115_Cell culture plate
116 Type I collagen solution
I17 Decompression container
lI8 Decompression pump
20 119 Saline for washing (PBS (-) )
120...Medium
12L.Hepatocyte
122...Hepatocyte spheroid
125a, 125b, 127a, 127b, 129a, 129b, 129c...Projection
25 arrangement pattern
1111...Non-slip portion
1112 Joint
1113 Cut face

53
e Claims
[Claim 1]
A culture substrate for culturing cells,
5 the culture substrate comprising a culture sheet, and a
culture sheet retaining member which retains the culture
sheet,
the culture sheet having a culture region, the culture
region having a plurality of projections formed therein, a
10 partition which partitions the culture region and is taller
than the projections being formed, and the constitutional
proportion of the projections in the culture region being
in the range from 20% to 75%.
20 [Claim 3]
The culture substrate according to claim 2, wherein
the frame is in contact with the culture sheet retaining
member.
25 [Claim 4]
54
tit The culture substrate according to claim 1, wherein
the sheet retaining member has at least one hole portion,
and the culture sheet is constituted at the bottom of the
hole portion.
S
[Claim 5J
Th~ cult~re ~upstrate according to claim 4, wherein
the sheet retaining member has a protrusion formed at the
bottom thereof, and the protrusion and the culture sheet
10 are welded.
[Claim 6 J
The culture substrate according to claim 2, wherein
the frame body has a square or round shape.
lS
[Claim 7 J
The culture substrate according to claim 4, wherein
the hole portion has a square or +ound sqape.
20 [Claim 8J
The culture substrate according to claim 1, wherein
the culture sheet has a plurality of the culture regions.
[Claim 9J
2S A culture sheet for culturing cells, the culture
55
tit sheet comprising a plurality of culture regions,
a plurality of projections formed in each of the culture
regions, and
a partition whiGP partitions the culture regions and is
5 taller than the projections, and the constitutional
proportion of the projections in the culture region being
in the range from 20% to 75%.
[Claim 10]
10 The culture sheet according to claim 9, wherein,
the culture region has a first region and a second region,
the width/diameter of the projections in the first region
and the width/diameter of the projections in the second
region are different.
15
The culture s~strate according to claim 1, wherein
the partition and a plurality of the projections in the
culture sheet are formed integrally from the same material.
20
[Claim 12]
The culture sheet according to claim 9, wherein,
the partition and a plurality of the projections are .formed
integrally from the same material.
25 [Claim 1$]
56
The culture substrate according to claim 1, wherein
the constitutional proportion of projections in the culture
regions is in the range from 40% to 50%.
~ [Cla;i..m 14]
The culture sheet according to claim 9, wherein,
the constitutional proportion of projections in the culture
regions is in the range from 40% to 50%.
10 (Claim 15]
The culture sheet according to claim 9, wherein,
projections having different constitutional proportions of
the projections ranging from 20% to 75% are arranged in
15
each o! the culture regions partitioned
Dated this zo" Dayof June 2013
by tile part~>_/
Of Anand AndAnand Advocates
Agentfor the Applicant