U.S. patent application number 14/906699 was filed with the patent office on 2016-06-23 for method for integrating biological tissues with a vascular system.
This patent application is currently assigned to PUBLIC UNIVERSITY CORPORATION YOKOHAMA CITY- UNIVERSITY. The applicant listed for this patent is PUBLIC UNIVERSITY CORPORATION YOKOHAMA CITY UNIVERSITY. Invention is credited to Yoshinobu TAKAHASHI, Takanori TAKEBE, Hideki TANIGUCHI.
Application Number | 20160177270 14/906699 |
Document ID | / |
Family ID | 52393202 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160177270 |
Kind Code |
A1 |
TAKEBE; Takanori ; et
al. |
June 23, 2016 |
METHOD FOR INTEGRATING BIOLOGICAL TISSUES WITH A VASCULAR
SYSTEM
Abstract
The present invention provides a method of constituting a tissue
construct in vitro using a tissue without depending on scaffold
materials. A method of integrating a biological tissue with a
vascular system in vitro, comprising coculturing a biological
tissue with vascular cells and mesenchymal cells. A biological
tissue which has been integrated with a vascular system by the
above-described method. A method of preparing a tissue or an organ,
comprising transplanting the biological tissue described above into
a non-human animal and differentiating the biological tissue into a
tissue or an organ in which vascular networks have been
constructed. A method of regeneration or function recovery of a
tissue or an organ, comprising transplanting the biological tissue
described above into a human or a non-human animal and
differentiating the biological tissue into a tissue or an organ in
which vascular networks have been constructed. A method of
preparing a non-human chimeric animal, comprising transplanting the
biological tissue described above into a non-human animal and
differentiating the biological tissue into a tissue or organ in
which vascular networks have been constructed. A method of
evaluating a drug, comprising using at least one member selected
from the group consisting of the biological tissue described above,
the tissue or organ prepared by the method described above, and the
non-human chimeric animal prepared by the method described above. A
composition for regenerative medicine, comprising a biological
tissue which has been integrated with a vascular system by the
method described above.
Inventors: |
TAKEBE; Takanori;
(Yokohama-shi, JP) ; TANIGUCHI; Hideki;
(Yokohama-shi, JP) ; TAKAHASHI; Yoshinobu;
(Yokohama-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PUBLIC UNIVERSITY CORPORATION YOKOHAMA CITY UNIVERSITY |
Yokohama-shi, Kanagawa |
|
JP |
|
|
Assignee: |
PUBLIC UNIVERSITY CORPORATION
YOKOHAMA CITY- UNIVERSITY
Yokohama--shi, Kanagawa
JP
|
Family ID: |
52393202 |
Appl. No.: |
14/906699 |
Filed: |
July 15, 2014 |
PCT Filed: |
July 15, 2014 |
PCT NO: |
PCT/JP2014/068808 |
371 Date: |
January 21, 2016 |
Current U.S.
Class: |
424/9.2 ;
424/93.7; 435/29; 435/347; 435/373 |
Current CPC
Class: |
A01K 2267/0331 20130101;
C12N 5/0691 20130101; A61K 35/44 20130101; A61L 27/3808 20130101;
A01K 67/0271 20130101; A61L 27/3834 20130101; G01N 33/5082
20130101; A01K 2267/035 20130101; C12N 2502/13 20130101; C07D
499/21 20130101; A01K 2267/0325 20130101; A01K 2207/12 20130101;
A61L 27/3886 20130101; A61K 35/28 20130101; A61K 49/0008 20130101;
A01K 2267/0362 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071; A61K 49/00 20060101 A61K049/00; A61K 35/28 20060101
A61K035/28; G01N 33/50 20060101 G01N033/50; A61K 35/44 20060101
A61K035/44 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 23, 2013 |
JP |
2013-153056 |
Claims
1. A method of integrating a biological tissue with a vascular
system in vitro, comprising coculturing a biological tissue with
vascular cells and mesenchymal cells.
2. The method of claim 1, wherein the biological tissue is
cocultured with vascular cells and mesenchymal cells without using
scaffold materials.
3. The method of claim 1 or 2, wherein by coculturing the
biological tissue with vascular cells and mesenchymal cells, the
biological tissue is integrated with a vascular system so that the
function of the biological tissue is maintained and/or
improved.
4. A biological tissue which has been integrated with a vascular
system by the method of claim 1.
5. A method of preparing a tissue or an organ, comprising
transplanting the biological tissue of claim 4 into a non-human
animal and differentiating the biological tissue into a tissue or
an organ in which vascular networks have been constructed.
6. A method of regeneration or function recovery of a tissue or an
organ, comprising transplanting the biological tissue of claim 4
into a human or a non-human animal and differentiating the
biological tissue into a tissue or an organ in which vascular
networks have been constructed.
7. A method of preparing a non-human chimeric animal, comprising
transplanting the biological tissue of claim 4 into a non-human
animal and differentiating the biological tissue into a tissue or
organ in which vascular networks have been constructed.
8. A method of evaluating a drug, comprising using at least one
member selected from the group consisting of the biological tissue
of claim 4, the tissue or organ prepared by the method of claim 5,
and the non-human chimeric animal prepared by the method of claim
7.
9. A composition for regenerative medicine, comprising the
biological tissue of claim 4.
10. The composition of claim 9, which is used for preparing a
tissue or an organ.
11. The composition of claim 9, which is used for regeneration or
function recovery of a tissue or an organ.
12. The composition of any one of claims 9 to 11, wherein the
biological tissue differentiates into a tissue or an organ with
vascular networks upon transplantation into a living body.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of biological
tissues with a vascular system. More specifically, the present
invention relates to a method of preparing three-dimensional
tissues with vascular networks from tissues induced from
pluripotent stem cells, etc. or tissues (such as normal or cancer
tissue) isolated from individuals.
BACKGROUND ART
[0002] Recently, the use of normal/cancer tissues isolated from
individuals or tissues induced from pluripotent stem cells has
attracted a great deal of attention as a way to realize drug
discovery screening to develop new pharmaceuticals, and
regenerative medicine to compensate for the functions of lost
organs.
[0003] As attempts to induce three-dimensional tissues from
pluripotent stem cells or the like, studies have been reported in
which spheroidal tissue fragments are formed and directed for cell
differentiation in such areas as the liver, pancreas or nerve
(Non-Patent Document No. 1: Takayama K, et al., Biomaterials. 2013
February; 34(7):1781-9; Non-Patent Document No. 2: Saito H, et al.,
PLoS ONE. 2011; 6(12): e28209; and Non-Patent Document No. 3:
Eiraku M, et al., Nature 2011, 472, 51-56). However, none of the
tissues induced by those methods have vasculatures. Vasculatures
have such a role that, once transplanted, they supply the tissues
with oxygen and nutrients that are necessary for their survival.
What is more, it is believed that, even before blood flows into the
tissue, recapitulating three-dimensional tissue structures with
blood vessels and cell polarity as well is important for the
differentiation, proliferation and maintenance of cells. Therefore,
avascular tissues not only fail to engraft upon transplantation and
suffer from inner necrosis, but also fail to achieve tissue
maturation that is associated with vascularization. It has,
therefore, been difficult for avascular tissues to exhibit adequate
functions.
[0004] Accordingly, for the purpose of integrating vasculatures to
a three-dimensional tissue, a method has been invented in which
tissues (such as pancreatic islets) isolated from individuals are
seeded on a carrier (scaffold material) and cocultured with
vascular endothelial cells, fibroblast cells, or the like
(Non-Patent Document No. 4: Kaufman-Francis K, et al., PLoS ONE
2012, 7(7): e40741).
[0005] However, this method has a limitation in spatial arrangement
caused by scaffold materials and cell behavior is greatly affected.
Therefore, it is difficult for this method to construct a precise
structure like a biological tissue and appropriate interactions
between cells are not recapitulated. Consequently, problems arise
such as inhibited maturation and proliferation of cells in tissues,
and delayed reconstitution of functional vascular networks that
leads to poor engraftment after transplantation. There is yet
another serious problem that may occur in transplantation and the
like; the scaffold material used causes a foreign-body reaction
which will result in inflammation or the like.
[0006] As described above, reconstitution of three-dimensional
tissues having vascular networks is desirable if applications in
industry and regenerative medicine are intended but, in fact, no
method is yet to be established that is capable of constituting a
tissue construct with vasculatures in vitro using a tissue without
depending on scaffold materials.
PRIOR ART LITERATURE
Non-Patent Documents
[0007] Non-Patent Document No. 1: Takayama K, et al., Biomaterials.
2013 February; 34(7):1781-9 [0008] Non-Patent Document No. 2: Saito
H, et al., PLoS ONE. 2011; 6(12): e28209 [0009] Non-Patent Document
No. 3: Eiraku M, et al., Nature 2011, 472, 51-56 [0010] Non-Patent
Document No. 4: Kaufman-Francis K, et al., PLoS ONE 2012, 7(7):
e40741
DISCLOSURE OF THE INVENTION
Problem for Solution by the Invention
[0011] For the realization of drug development and regenerative
medicine for diseases in the liver, pancreas, kidney, intestine,
lung, etc., it is essential to recapitulate a three-dimensional
tissue structure associated with vascularization, as well as cell
polarity. Briefly, in order to maximize the function of a tissue
induced from pluripotent stem cells or a tissue isolated from an
individual, it is necessary to form a three-dimensional tissue
construct that enables reconstitution of vascular networks.
[0012] In this connection, the present inventors have established
an innovative three-dimensional culture technique which realized
"directed differentiation of organ cells based on organ
reconstitution", by utilizing spatiotemporal interactions between
different cell lineages (Nature, 499:481-484, 2013; WO2013/047639
titled "Method for Producing Tissue and Organ"). Briefly, by
recapitulating those intracellular interactions among organ cells,
vascular cells and mesenchymal cells which are essential for early
processes of organogenesis, a primordium of steric organ (an organ
bud) is induced, thus providing a platform for enabling the
generation of functional organs with vascular networks. However,
this method starts with organ cells and it has not been elucidated
as to whether a primordium of three-dimensional tissue with
vascular networks can be generated by using a tissue
fragment(tissue).
[0013] The present invention aims at providing a method of
constituting a tissue construct with vasculatures in vitro from a
tissue without depending on scaffold materials.
Means to Solve the Problem
[0014] The present inventors have found that close intercellular
reactions between organ cells (from which organs develop) and
vascular endothelial cells/mesenchymal cells direct the progress of
steric tissue formation that involves autonomous tissue structure
constitution and cell differentiation (Nature, 499:481-484, 2013;
WO2013/047639 titled "Method for Producing Tissue and Organ").
However, it is yet to be made clear if vascular networks can be
integrated into tissue fragments.
[0015] The present invention attempts to artificially generate
steric tissues having vascular networks in vitro starting with
tissues by artificially recapitulating such early processes of
organogenesis. Further, by transplanting the steric tissues into
living bodies, the present invention intends to create a
vascularized steric tissue which, when transplanted into a living
body after being induced in a culture system, restarts blood flow
to enable the tissue function to achieve maturation and
maintenance.
[0016] The present inventors have cocultured tissues isolated from
individuals (up to approximately 10-3,000 .mu.m) or tissues induced
from pluripotent stem cells (up to approximately 10-3,000 .mu.m)
with vascular cells and mesenchymal cells at appropriate mixing
ratios. The methods described below were used for inducing steric
tissues.
1. Three-dimensional tissues are formed by coculturing tissues with
vascular/mesenchymal cells on a carrier such as Matrigel. 2.
Three-dimensional tissues are formed by coculturing tissues with
vascular/mesenchymal cells on a plate of such a shape that cells
gather in the bottom.
[0017] By culturing tissues for a short period according to the
above-described methods, it was possible to induce in vitro steric
tissues integrated with microvasculatures.
[0018] Further, the present inventors successfully created
tissues/organs with a highly ordered tissue structure comparable to
that of adult tissues; when the steric tissues induced in a culture
system were by transplanted into living bodies, reconstruction of
functional vascular networks was induced, whereupon blood perfusion
was restarted to create the above-described tissues/organs.
[0019] This technique of attempting three-dimensional
reconstitution of tissues/organs based on the induction of
self-organization from tissues through intercellular interactions
was not available in the past and is believed to provide a method
whose novelty is extremely high.
[0020] A summary of the present invention is as described below.
[0021] (1) A method of integrating a biological tissue with a
vascular system in vitro, comprising coculturing a biological
tissue with vascular cells and mesenchymal cells. [0022] (2) The
method of (1) above, wherein the biological tissue is cocultured
with vascular cells and mesenchymal cells without using scaffold
materials. [0023] (3) The method of (1) or (2) above, wherein by
coculturing the biological tissue with vascular cells and
mesenchymal cells, the biological tissue is integrated with a
vascular system so that the function of the biological tissue is
maintained and/or improved. [0024] (4) A biological tissue which
has been integrated with a vascular system by the method of any one
of (1) to (3) above. [0025] (5) A method of preparing a tissue or
an organ, comprising transplanting the biological tissue of (4)
above into a non-human animal and differentiating the biological
tissue into a tissue or an organ in which vascular networks have
been constructed. [0026] (6) A method of regeneration or function
recovery of a tissue or an organ, comprising transplanting the
biological tissue of (4) above into a human or a non-human animal
and differentiating the biological tissue into a tissue or an organ
in which vascular networks have been constructed. [0027] (7) A
method of preparing a non-human chimeric animal, comprising
transplanting the biological tissue of (4) above into a non-human
animal and differentiating the biological tissue into a tissue or
organ in which vascular networks have been constructed. [0028] (8)
A method of evaluating a drug, comprising using at least one member
selected from the group consisting of the biological tissue of (4)
above, the tissue or organ prepared by the method of (5) above, and
the non-human chimeric animal prepared by the method of (7) above.
[0029] (9) A composition for regenerative medicine, comprising the
biological tissue of (4) above. [0030] (10) The composition of (9)
above, which is used for preparing a tissue or an organ. [0031]
(11) The composition of (9) above, which is used for regeneration
or function recovery of a tissue or an organ. [0032] (12) The
composition of any one of (9) to (11) above, wherein the biological
tissue differentiates into a tissue or an organ with vascular
networks upon transplantation into a living body.
[0033] According to the present invention, normal/cancer tissues
isolated from individuals or tissues induced from pluripotent stem
cells are cocultured with vascular cells and mesenchymal cells
under appropriate environments, whereby it has become possible to
constitute steric tissue constructs in vitro that are integrated
with vascular networks. Since vascular networks which are essential
for maturation, maintenance, repair, etc. of tissues are provided,
highly functional tissues are reconstituted, potentially providing
a platform for preparing tissue constructs useful for drug
discovery screening and regenerative medicine.
[0034] Conventionally, tissue constructs obtained from pluripotent
stem cells by directed differentiation remained less mature in the
differentiation stage than functional cells that constitute adult
tissues. This is because terminal differentiation of functional
cells has not been achieved by the conventional directed
differentiation method.
[0035] According to the present invention, it has become possible
to reconstitute a tissue integrated with vascular networks and one
may expect that a method of directing terminal differentiation of
human functional cells will be established (for example,
reconstitution of cell polarity in vasculature); hence, the present
invention is highly valuable as a technique for creating human
functional cells.
[0036] On the other hand, the tissues derived from organs removed
from individuals markedly deteriorate in function immediately after
they are isolated and it has been difficult to maintain their
functions. If an improvement/maintenance of a tissue's function is
achieved by integrating vascular networks to it according to the
present invention, it may be possible to provide a transplantation
technique with remarkable therapeutic efficacy for those patients
who have not benefited adequately from the conventional tissue
transplantation therapies for the reason that the transplant has no
vascular system (e.g., islet transplantation therapy). Further, it
will become possible to maximize the functions of various organs in
vitro or in vivo and one may expect that the present invention will
provide a platform useful for drug discovery screening.
[0037] Further, according to the present invention, it is possible
to reconstitute a steric human tissue construct having a vascular
system. Therefore, it will become possible to generate a tissue or
an organ that permits a blood flow in an appropriately arranged
vascular system and which has been entirely unachievable by
conventional techniques. Consequently, one may expect that the
present invention will provide a completely novel analysis system
for evaluating the efficacy of pharmaceuticals by which the
relationship between development of drug efficacy and blood vessels
and other factors that have been difficult to analyze by existing
evaluation systems can be evaluated.
[0038] Further, the advantages the present invention have over the
previously disclosed method (Nature, 499:481-484, 2013;
WO2013/047639) in which close intercellular reactions between organ
cells and vascular endothelial cells/mesenchymal cells are relied
upon to direct the progress of steric tissue formation that
involves autonomous tissue structure constitution and cell
differentiation may be enumerated as follows.
1. It is possible to provide a vascular system even for those
tissues which are constituted from difficult-to-expand cells (such
as pancreatic .beta. cells, renal glomerular epithelial/renal
tubular epithelial cells, hepatic cells, intestinal epithelial
cells, alveolar epithelial cells, tumor cells, trophectodermal
cells, iPS cell-derived endodermal cells, iPS cell-derived
mesodermal cells, iPS cell-derived from ectodermal cells and iPS
cell-derived tissue stem/progenitor cells) and examples of such
tissues include pancreatic islets, renal glomeruli, liver tissues,
intestinal crypts, pulmonary alveoli, tumor tissues,
trophectodermal tissues, iPS cell-derived endodermal cell-derived
spheroids, iPS cell-derived mesodemial cell-derived spheroids, iPS
cells-derived ectodermal cell-derived spheroids and iPS
cell-derived tissue stem/progenitor cell-derived spheroids. 2. It
is possible to provide a vascular system for larger tissues.
Tissues can be generated by the method disclosed in Nature,
499:481-484, 2013; WO2013/047639 only in the case where isolated
cells are used. The method of the present invention has been
confirmed to be capable of integrating a vascular system for
tissues, rather than cells, that are approximately 10-3,000 .mu.m
in size. 3. By integrating a vascular system for a tissue fragment
derived from stem cells such as iPS cells, it is possible to
recapitulate environments which are similar to the developmental
processes of biological tissues and directed differentiation into
functional cells that constitute a tissue of interest can be
achieved efficiently.
Effect of the Invention
[0039] According to the present invention, normal or cancer tissues
isolated from individuals or tissues induced from pluripotent stem
cells or the like are cocultured with vascular cells and
mesenchymal cells, whereby it has become possible to constitute
steric tissue constructs in vitro that are integrated with vascular
networks. This technique is applicable to, for example, generation
of human functional cells; organ transplantation; drug discovery
screening; novel analysis systems to evaluate the relationship
between development of drug efficacy and blood vessels.
[0040] The present specification encompasses the contents disclosed
in the specification and/or drawings of Japanese Patent Application
No. 2013-153056 based on which the present application claims
priority.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1A This figure shows the integration of vascular
networks to pancreatic islet (hereinafter, frequently referred to
simply as "islet") tissues.
A) Validation of media for culturing mouse islets using
Live/Dead.TM. Cell Imaging Kit (green: viable cells, red: dead
cells).
[0042] FIG. 1B This figure shows the integration of vascular
networks to islet tissues.
B) Quantification data for A).
[0043] FIG. 1C This figure shows the integration of vascular
networks to islet tissues.
C) Time-lapse imaging of three-dimensional tissue constituting
processes using mouse islets (colorless), vascular endothelial
cells (green) and mesenchymal stem cells (red).
[0044] FIG. 1DE This figure shows the integration of vascular
networks to islet tissues.
D) Mouse islets at 24 hours of culture. E) Mouse islets, vascular
endothelial cells and mesenchymal stem cells at 24 hours of
coculture. E') Immunohistological analysis of the three-dimensional
tissue generated in E) (green: insulin, red: human CD31).
[0045] FIG. 1F This figure shows the integration of vascular
networks to islet tissues.
F) Determination of survival or death of mouse islet cells using
Live/Dead.TM. Cell Imaging Kit (green: viable cells, red: dead
cells).
[0046] FIG. 1G This figure shows the integration of vascular
networks to islet tissues.
G) Quantification data for F).
[0047] FIG. 1H This figure shows the integration of vascular
networks to islet tissues.
H) Increase of insulin concentration released from cocultured mouse
islets.
[0048] FIG. 1I This figure shows the integration of vascular
networks to islet tissues.
I) Glucose tolerance test in vitro.
[0049] FIG. 1J-1 This figure shows the integration of vascular
networks to islet tissues.
J-1) Group of genes whose expressions are markedly enhanced by
coculture with vascular endothelial cells and mesenchymal stem
cells.
[0050] FIG. 1J-2 This figure shows the integration of vascular
networks to islet tissues.
J-2) Continuation from J-1).
[0051] FIG. 1J-3 This figure shows the integration of vascular
networks to islet tissues.
J-3) Continuation from J-2).
[0052] FIG. 2A This figure shows preparation of vascularized islet
fragments.
A) Autonomous formation of vascularized islet fragments using a
culture plate of such a shape that cells gather in the bottom
(vascularized tissues are formed even when the number of mouse
islets is changed).
[0053] FIG. 2B This figure shows preparation of vascularized islet
fragments.
B) Reviewing the conditions of vascular endothelial cell number and
mesenchymal stem cell number.
[0054] FIG. 2C This figure shows preparation of vascularized islet
fragments.
C) Time-lapse imaging of the processes of formation of vascularized
islet fragments (changes in cell morphology caused by coculture;
mouse islets: blue; vascular endothelial cells: green; mesenchymal
stem cells: red).
[0055] FIG. 2D This figure shows preparation of vascularized islet
fragments.
D) Prepared vascularized islet fragments; mouse islets (red),
vascular endothelial cells (green) and mesenchymal stem cells
(colorless).
[0056] FIG. 2E This figure shows preparation of vascularized islet
fragments.
E) Histological analysis of vascularized islet fragments; mouse
islets (red), vascular endothelial cells (green), mesenchymal stem
cells (colorless) and mouse CD31 (blue).
[0057] FIG. 3AB This figure shows validation of function upon
transplantation of vascularized tissue.
A) Macroimaging of the site of transplantation of vascularized
islets (yellow arrow indicates blood inflow). B) Macroimaging of
the site of transplantation of islets alone (control group).
[0058] FIG. 3CD This figure shows validation of function upon
transplantation of vascularized tissue.
C) Blood perfusion into vascularized islets; mouse islets (green),
vascular endothelial cells (colorless), mesenchymal stem cells
(colorless), dextran (red). D) Blood perfusion around transplanted
islets; mouse islets (green), vascular endothelial cells
(colorless), mesenchymal stem cells (colorless), dextran (red).
[0059] FIG. 3E This figure shows validation of function upon
transplantation of vascularized tissue.
E) Transplantation of vascularized islets into the subcapsular
space of the kidney using diabetes model mice; blood glucose
transition.
[0060] FIG. 3F This figure shows validation of function upon
transplantation of vascularized tissue.
F) Blood glucose transition in diabetes model mice.
[0061] FIG. 3G This figure shows validation of function upon
transplantation of vascularized tissue.
G) Body weight transition in diabetes model mice.
[0062] FIG. 3H This figure shows validation of function upon
transplantation of vascularized tissue.
H) Survival ratios in diabetes model mice.
[0063] FIG. 3I This figure shows validation of function upon
transplantation of vascularized tissue.
I) In vivo glucose tolerance test.
[0064] FIG. 3JK This figure shows validation of function upon
transplantation of vascularized tissue.
J) Histological analysis of vascularized islets transplanted into
CW. K) Histological analysis of islets transplanted into CW.
[0065] FIG. 3L This figure shows validation of function upon
transplantation of vascularized tissue.
L) Histological analysis of vascularized islets transplanted into
the subcapsular space of the kidney; insulin (green), laminin
(red), DAPI (blue).
[0066] FIG. 3M This figure shows validation of function upon
transplantation of vascularized tissue.
M) Histological analysis of islets transplanted into the
subcapsular space of the kidney; insulin (green), laminin (red),
DAPI (blue).
[0067] FIG. 4 This figure shows the integration of vascular
networks to renal glomeruli.
A) Autonomous formation of a three-dimensional tissue derived from
mouse renal glomeruli, vascular endothelial cells and mesenchymal
stem cells using a 24-well dish. B) Autonomous formation of a
three-dimensional tissue derived from mouse renal glomeruli,
vascular endothelial cells and mesenchymal stem cells using a
culture plate (substrate?) of such a shape that cells gather in the
bottom (time-lapse imaging of the three-dimensional tissue using
mouse renal glomeruli (green), vascular endothelial cells (red) and
mesenchymal stem cells (blue)). C) Macroscopic image of
vascularized three-dimensional mouse renal glomerular tissue at 24
hours of culture using a 24-well dish. D) Macroscopic image of
vascularized three-dimensional mouse renal glomerular tissue at 24
hours of culture using a 96-well dish. E) Confirmation of
vascularization and engraftment at the site of transplantation of
vascularized renal glomeruli. F) Live imaging of the site of
transplantation of vascularized renal glomeruli (mouse renal
glomeruli (red), human vascular endothelial cells (green), mouse
vascular endothelial cells (blue)).
[0068] FIG. 5 This figure shows the integration of vascular
networks to tumor tissues.
A) Autonomous formation of a three-dimensional tissue derived from
human pancreatic tumor tissue (red), vascular endothelial cells
(green) and mesenchymal stem cells (colorless) using a 24-well
dish. B) Lapse imaging of a three-dimensional tissue formed
autonomously from mouse pancreatic cancer tissue, vascular
endothelial cells and mesenchymal stem cells at 24 hours of culture
using a 24-well dish. C) Enhanced expression of a cancer stem cell
marker (CD44) by formation of vascularized tissue.
[0069] FIG. 6 This figure shows the integration of vascular
networks to liver tissues.
A) Time-lapse imaging of the process of formation of a
three-dimensional tissue derived from mouse liver tissues (green),
vascular endothelial cells (red) and mesenchymal stem cells
(colorless). B) Autonomous formation of a three-dimensional tissue
derived from mouse liver tissues (green), vascular endothelial
cells (red) and mesenchymal stem cells (colorless) using a culture
plate (substrate?) of such a shape that cells gather in the bottom.
C) Macroimaging of the site of transplantation of vascularized
liver tissues. D) Reconstitution of a vascular system inside the
vascularized liver tissues.
[0070] FIG. 7 This figure shows the integration of vascular
networks to intestinal tissues.
A) Time-lapse imaging of the process of formation of a
three-dimensional tissue using mouse intestinal tissues (red),
vascular endothelial cells (green) and mesenchymal stem cells
(colorless). B) Autonomous formation of a three-dimensional tissue
derived from intestinal tissues (red), vascular endothelial cells
(green) and mesenchymal stem cells (colorless) using a culture
plate (substrate?) of such a shape that cells gather in the bottom.
C) Macroimaging of the site of transplantation of vascularized
intestinal tissues. D) In vivo live imaging of the site of
transplantation of vascularized intestinal tissues (mouse
intestinal tissues (red), vascular endothelial cells (green) and
mesenchymal stem cells (colorless)).
[0071] FIG. 8 This figure shows the integration of vascular
networks to pulmonary tissues.
A) Autonomous formation of a three-dimensional tissue using mouse
pulmonary tissues (red), vascular endothelial cells (green) and
mesenchymal stem cells (colorless). B) Macroimaging of the site of
transplantation of vascularized pulmonary tissue. C) In vivo live
imaging of the site of transplantation of vascularized pulmonary
tissue (mouse pulmonary tissues (red), vascular endothelial cells
(green), mesenchymal stem cells (colorless) and mouse CD31
(blue)).
[0072] FIG. 9 This figure shows the integration of vascular
networks to iPS cell-derived endodermal tissues.
A) Outline of the method of application to human iPS cell-derived
endodermal cell spheroids. B) Autonomous formation of a
three-dimensional tissue using human iPS cell-derived endodermal
tissue fragments, vascular endothelial cells and mesenchymal stem
cells. C) Fluorescent image observation of a three-dimensional
tissue constituted from human iPS cell-derived endodermal tissue
fragments (colorless), vascular endothelial cells (red) and
mesenchymal stem cells (colorless).
BEST MODES FOR CARRYING OUT THE INVENTION
[0073] Hereinbelow, the present invention will be described in
detail.
[0074] The present invention provides a method of integrating a
vascular system for a biological tissue in vitro, comprising
coculturing a biological tissue with vascular cells and mesenchymal
cells.
[0075] In the present specification, the term "biological tissue"
refers to a construct constituted from a plurality of cells. For
example, normal/abnormal tissues or cancer tissues isolated from
individuals as well as tissues induced from pluripotent stem cells
(such as induced pluripotent stem cells (iPS cells) and embryonic
stem cells (ES cells)), tissue stem/progenitor cells,
differentiated cells or the like may be enumerated. As biological
tissues, those derived from humans may primarily be used.
Biological tissues derived from non-human animals (e.g., animals
used, for example, as experimental animals, pet animals, working
animals, race horses or fighting dogs; more specifically, mouse,
rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken,
shark, devilfish, ratfish, salmon, shrimp, crab or the like) may
also be used.
[0076] In the present specification, the term "vascular system"
refers to a structure composed of vascular endothelial cells and
its supporting cells. Vascular systems not only mainain tissues but
also play an important role in the maturation process of tissues.
Vascular structures have such a role that, once transplanted, they
supply the tissues with oxygen and nutrients that are necessary for
their survival. What is more, it is believed that even before blood
flows into the tissue, recapitulating three-dimensional tissue
structures with blood vessels and cell polarity as well is
important for the differentiation, proliferation and maintenance of
cells. Therefore, avascular tissues not only fail to engraft upon
transplantation and suffer from inner necrosis, but also fail to
achieve tissue maturation that is associated with vascularization.
It has, therefore, been difficult for avascular tissues to exhibit
adequate functions.
[0077] In the present specification, the terms "integrating a
vasculature system" and "vascularization" mean that a vascular
system composed of vascular endothelial cells and its supporting
cells is integrated directly with a target tissue. When a
biological tissue integrated with a vascular system is transplanted
into a living body, maturation of blood vessels is observed and
upon connecting to the host blood vessels, blood perfusion starts,
enabling induction to a functional tissue/organ having vascular
networks.
[0078] Vascular cells may be isolated from vascular tissues but
they are in no way limited to those isolated therefrom. Vascular
cells may be derived from totipotent or pluripotent cells (such as
iPS cells and ES cells) by induction of differentiation. As
vascular cells, vascular endothelial cells are preferable. In the
present specification, the term "vascular endothelial cells" means
cells constituting vascular endothelium or cells capable of
differentiating into such cells (for example, vascular endothelial
progenitor cells and vascular endothelial stem cells). Whether a
cell is vascular endothelial cell or not can be determined by
checking to see if they express marker proteins such as TIE2,
VEGFR-1, VEGFR-2, VEGFR-3 and CD31 (if any one or more of the
above-listed marker proteins are expressed, the cell can safely be
regarded as a vascular endothelial cell). Further, as markers for
vascular endothelial progenitor cells, c-kit, Sca-1, etc. have been
reported. If these markers are expressed, the cell of interest can
be confirmed as a vascular endothelial progenitor cell (S. Fang, et
al., PLOS Biology, 2012; 10(10): e1001407). Among the terms used by
those skilled in the art, the following are included in the
"vascular endothelial cell" of the present invention: endothelial
cells, umbilical vein endothelial cells, endothelial progenitor
cells, endothelial precursor cells, vasculogenic progenitors,
hemangioblast (H J. Joo, et al. Blood. 25; 118(8):2094-104 (2011))
and so on. As vascular cells, human-derived cells are mainly used.
However, vascular cells derived from non-human animals (e.g.,
animals used, for example, as experimental animals, pet animals,
working animals, race horses or fighting dogs; more specifically,
mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep,
chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the
like) may also be used. Vascular cells may be obtained from cord
blood, umbilical cord vessels, neonatal tissues, liver, aorta,
brain, bone marrow, adipose tissues, and so forth.
[0079] In the present invention, the term "mesenchymal cells" means
connective tissue cells that are mainly located in mesodemi-derived
connective tissues and which form support structures for cells that
function in tissues. The "mesenchymal cell" is a concept that
encompasses those cells which are destined to, but are yet to,
differentiate into mesenchymal cells. Mesenchymal cells to be used
in the present invention may be either differentiated or
undifferentiated. Preferably, undifferentiated mesenchymal cells
are used. Whether a cell is an undifferentiated mesenchymal cell or
not may be confirmed by checking to see if the cell expresses
marker proteins such as Stro-1, CD29, CD44, CD73, CD90, CD105,
CD133, CD271 or Nestin (if any one or more of the above-listed
marker proteins are expressed, the cell can safely be regarded as
an undifferentiated mesenchymal cell). A mesenchymal cell in which
none of the above-listed markers is expressed can be judged as
differentiated mesenchymal cell. Among the terms used by those
skilled in the art, the following are included in the "mesenchymal
cell" of the present invention: mesenchymal stem cells, mesenchymal
progenitor cells, mesenchymal cells (R. Peters, et al. PLoS One.
30; 5(12):e15689 (2010)) and so on. As mesenchymal cells,
human-derived cells are mainly used. However, mesenchymal cells
derived from non-human animals (e.g., animals used, for example, as
experimental animals, pet animals, working animals, race horses or
fighting dogs; more specifically, mouse, rat, rabbit, pig, dog,
monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish,
salmon, shrimp, crab or the like) may also be used.
[0080] The size of a biological tissue to be cocultured with
vascular cells and mesenchymal cells may be approximately 10-500
.mu.m, but is not limited to this range. Preferably, the size is
approximately 100-300 .mu.m. More preferably, the size is
approximately 100-150 .mu.m.
[0081] The numbers of vascular cells and mesenchymal cells to be
used for coculture may each be about
2.times.10.sup.2-1.times.10.sup.5 cells, preferably, about
2.times.10.sup.2-5.times.10.sup.4 cells, and more preferably, about
1.times.10.sup.4 cells, per biological tissue of approx. 150 .mu.m
in size.
[0082] The culture ratio of vascular cells and mesenchymal cells in
coculture is not particularly limited if it is within such a range
that a vascular system is provided for biological tissues. A
preferable cell count ratio as expressed by the vascular cell to
mesenchymal cell is 10-3:3-1.
[0083] The number of biological tissues in coculture is not
particularly limited if it is within such a range that a vascular
system is provided for biological tissues. Preferably, 1-100
tissues approx. 100-150 .mu.m in diameter are used for a mixture of
1.times.10.sup.4 vascular cells and 1.times.10.sup.4 mesenchymal
cells.
[0084] Either one or both of vascular cells and mesenchymal cells
may be substituted by substances such as factors secreted by
vascular cells, factors secreted by mesenchymal cells, and factors
secreted as a result of the presence of both vascular cells and
mesenchymal cells.
[0085] Examples of the substances such as factors secreted by
vascular cells, factors secreted by mesenchymal cells, and factors
secreted as a result of the presence of both vascular cells and
mesenchymal cells include, but are not limited to, FGF2, FGF5,
BMF4, BMP6, CTGF, angiopoietin 2, chemokine (C-C motif) ligand 14
and von Willebrand factor.
[0086] With respect to the amount of addition of these substances,
FGF2 may be added at 10-100 ng/ml, preferably at about 20 ng/ml,
per 1.times.10.sup.6 cells; and BMF4 may be added at 10-100 ng/ml,
preferably at about 20 ng/ml, per 1.times.10.sup.6 cells.
[0087] The medium used for culturing is not particularly limited.
Any medium may be used as long as it enables the integration of a
vascular system for biological tissues. Preferably, a medium for
culturing vascular cells (in particular, vascular endothelial
cells), a medium for culturing biological tissues or a mixture of
these two media may be used. As a medium for culturing vascular
cells (in particular, vascular endothelial cells), any medium may
be used but, preferably, a medium containing at least one of the
following substances may be used: hEGF (recombinant human
epithelial growth factor), VEGF (vascular endothelial growth
factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS,
antibiotics (e.g., gentamycin or amphotericin B), heparin,
L-glutamine, phenol red and BBE. As a medium for culturing vascular
endothelial cells, EGM-2 BulletKit (Lonza), EGM BulletKit (Lonza),
VascuLife EnGS Comp Kit (LCT), Human Endothelial-SFM Basal Growth
Medium (Invitrogen), human microvascular endothelial cell growth
medium (Toyobo) or the like may be used. The medium used for
culturing biological tissues is not particularly limited but, as a
medium for culturing islet tissues, RPMI1640 (Wako) or EGM.TM.
BulletKit.TM. (Lonza CC-4133) supplemented with 10% fetal bovine
serum (BWT Lot.S-1560), 20 mmol/L L-glutamine (Gibco) and 100
.mu.g/ml penicillin/streptomycin (Gibco) may preferably be used; as
a medium for culturing renal tissues (such as renal glomeruli),
RPMI1640 (Wako) supplemented with 20% fetal bovine serum (BWT
Lot.S-1560), 100 .mu.g/ml penicillin/streptomycin (Gibco) and
Insulin-Transferrin-SeleniumX (Gibco) may preferably be used; as a
medium for culturing intestinal tissues (such as crypt fragments),
RPMI1640 (Wako) supplemented with 20% fetal bovine serum (BWT
Lot.S-1560), 100 .mu.g/ml penicillin/streptomycin (Gibco) and
Insulin-Transferrin-SeleniumX (Gibco) may preferably be used; as a
medium for culturing liver tissues, DMEM/F12 (Invitrogen)
supplemented with 10% fetal bovine serum (ICN Lot.7219F), 2 mmol/L
L-glutamine (Gibco), 100 .mu.g/mL penicillin/streptomycin (Gibco),
10 mmol/L nicotinamide (Sigma), 50 .mu.mol/L 2-Mercaptoethanol,
1.times.10.sup.-7 mol/L 6.5% dexamethasone (Sigma),
2.6.times.10.sup.-4 M L-Ascorbic acid 2-phosphate sesquimagnesium
salt hydrate (Sigma), 5 mmol/L HEPES (Dojindo), 1 .mu.g/mL Human
recombinant insulin expressed in yeast (Wako), 50 ng/mL Human
recombinant HGF expressed in Sf21 insect cells (Sigma) and 20 ng/mL
Mouse Submaxillary Glands EGF (Sigma) may preferably be used; as a
medium for iPS cell-derived endodermal tissues, RPMI1640 (Wako)
supplemented with 1% B27 SUPPLEMENT X50 (Invitrogen 17504-044), 10
nG/ML BFGF Recombinant Human (Wako 060-04543) and 20 nG/ML BMP4
Recombinant Human (R&D 314-BP) may preferably be used; as a
medium for iPS cell-derived hepatic endodermal tissues, a medium
kit for sole use with hepatocytes (HCM.TM. BulletKit.TM. lonza
CC3198) freed of hEGF (recombinant human epithelial growth factor)
and supplemented with 0.1 .mu.M Dexamethasone (Sigma-Aldrich), 10
ng/ml Oncostatin M (R&D) and 10 ng/ml HGF (PromoKine) may
preferably be used; and as a medium for cancer tissues or pulmonary
tissues, the same media as that for vascular cells may preferably
be used.
[0088] Preferably, biological tissues are seeded on a substrate
such as gel and cocultured with vascular cells and mesenchymal
cells. The substrate may be a base material having a stiffness of
0.5-25 kPa. Examples of such base material include, but are not
limited to, gels (e.g., ranging from a stock solution to a 4-fold
dilution of Matrigel.TM., agarose gel, acrylamide gel, hydrogel,
collagen gel or urethane gel).
[0089] Alternatively, biological tissues may be cocultured with
vascular cells and mesenchymal cells on a plate of such a shape
that cells gather in the bottom. The plate used for this purpose is
not particularly limited as long as it has such a shape that cells
gather in the bottom. For example, PrimeSurface.TM. 96-well U plate
(Sumitomo Bakelite) may be used.
[0090] The temperature at the time of culture is not particularly
limited but it is preferably 30-40.degree. C., more preferably
37.degree. C.
[0091] The time period of culture is not particularly limited but
it is preferably 12-144 hours. For vascularization of adult tissues
such as islets, the culture period is more preferably about 12-24
hours. For vascularization of iPS cell-derived tissues, the culture
period is more preferably about 48-72 hours. For vascularization of
cancer tissues, the culture period is more preferably about 12-72
hours.
[0092] The biological tissue that has been integrated with a
vascular system by the method of the present invention may be a
construct characterized in that the complex tissue is autonomously
formed by cells or tissues. Further, the biological tissue that has
been integrated with a vascular system by the method of the present
invention may be a complex tissue in which the vascular system
directly integrates with (i.e., adheres to, connects to, or
continues to) the tissue.
[0093] In the method of the present invention, it is possible to
provide a vascular system for a biological tissue by coculturing
the biological tissue with vascular cells and mesenchymal cells
without using scaffold materials.
[0094] When a vascular system is provided for a biological tissue
by coculturing the biological tissue with vascular cells and
mesenchymal cells, the function of the biological tissue can be
maintained and/or improved. In addition to the maintenance and
improvement of the function of the biological tissue,
transplantation efficiency is sufficiently improved to provide a
treatment method having remarkable therapeutic effects.
[0095] Further, the present invention which enables reconstruction
of a vascular system will leads to the establishment of a method by
which terminally differentiated cells can be efficiently induced
from tissues derived from pluripotent stem cells such as iPS cells
and ES cells.
[0096] The biological tissue that has been integrated with a
vascular system by the method of the present invention may be a
complex tissue whose vascular system is capable of rapidly
functioning in vivo. Briefly, when the biological tissue integrated
with a vascular system by the method of the present invention is
transplanted into a living body (host), the time it takes for
anastomosis to host vessels to occur and for blood to flow in can
be greatly shortened, compared to cases where scaffold materials
are used [for example, when scaffold materials are used, 12 days
are taken (Engineered blood vessel networks connect to host
vasculature via wrapping-and-tapping anastomosis. Blood. 2011 Oct.
27; 118(17):4740-9) whereas the method of the present invention
takes only 1 to 2 days (see Examples described later)].
[0097] When the biological tissue integrated with a vascular system
by the method of the present invention is transplanted into a
non-human animal, vascular networks are constructed in the
transplanted tissue and blood perfusion starts to enable the
creation of a tissue or an organ having a highly ordered tissue
structure. Therefore, the present invention provides a method of
preparing a tissue or an organ, comprising transplanting a human or
a non-human animal with a biological tissue that has been
integrated with a vascular system by coculturing with vascular
cells and mesenchymal cells, and differentiating the biological
tissue into a tissue or an organ in which vascular networks have
been constructed. Non-human animals to be used in this method
include, but are not limited to, animals used, for example, as
experimental animals, pet animals, working animals, race horses or
fighting dogs; more specifically, mouse, rat, rabbit, pig, dog,
monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish,
salmon, shrimp, crab or the like may be used. Further, in order to
avoid immunorejection, the non-human animal to be used herein is
preferably an immunodeficient animal.
[0098] The site of transplantation of the biological tissue
integrated with a vascular system may be any site as long as
transplantation is possible. Specific examples of the
transplantation site include, but are not limited to, the
intracranial space, the mesentery, the liver, the spleen, the
kidney, the subcapsular space of the kidney, and the supraportal
space. When the biological tissue is to be transplanted into the
intracranial space, about 1 to 12 biological tissues of 500 .mu.m
in size, prepared in vitro, may be transplanted. When the
biological tissue is to be transplanted into the mesentery, about 1
to 12 biological tissues of 3-8 mm in size, prepared in vitro, may
be transplanted. When the biological tissue is to be transplanted
into the supraportal space, about 1 to 12 biological tissues of 3-8
mm in size, prepared in vitro, may be transplanted. When the
biological tissue is to be transplanted into the subcapsular space
of the kidney, about 1 to 6 biological tissues of 3-8 mm in size,
prepared in vitro, may be transplanted. When the biological tissue
is to be transplanted into the liver, spleen, kidney, lymph node or
blood vessel, about 100-2000 biological tissues of 100-200 .mu.m in
size, prepared in vitro, may be transplanted.
[0099] The tissues and organs prepared as described above may be
used in drug discovery screening and regenerative medicine.
[0100] Thus, the present invention provides a method of
regeneration or function recovery or a tissue or an organ,
comprising transplanting a human or a non-human animal with a
biological tissue that has been integrated with a vascular system
by coculturing with vascular cells and mesenchymal cells into, and
differentiating the biological tissue into a tissue or an organ in
which vascular networks have been constructed. Non-human animals to
be used in this method include, but are not limited to, animals
used, for example, as experimental animals, pet animals, working
animals, race horses or fighting dogs; more specifically, mouse,
rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken,
shark, devilfish, ratfish, salmon, shrimp, crab or the like may be
used.
[0101] Further, the present invention provides a composition for
regenerative medicine, comprising a biological tissue that has been
integrated with a vascular system by coculturing with vascular
cells and mesenchymal cells.
[0102] The composition of the present invention can be transplanted
into a living body to prepare a tissue or an organ. The composition
of the present invention can also be transplanted into a living
body to regenerate a tissue or an organ or recover its function. As
the living body, not only humans but also animals (such as ones
used as experimental animals, pet animals, working animals, race
horses or fighting dogs; more specifically, mouse, rat, rabbit,
pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish,
ratfish, salmon, shrimp, crab or the like) may be used.
[0103] After the composition of the present invention is
transplanted into a living body, the biological tissue is capable
of differentiating into a tissue or an organ having vascular
networks. In the vascular networks, blood perfusion can occur. It
is believed that the occurrence of blood perfusion in the vascular
networks enables generation of a tissue or an organ having a highly
ordered tissue structure either comparable or nearly comparable to
the tissue structure of adult tissues.
[0104] The composition of the present invention may contain
additives including, for example, tissue vascularization promoters
such as FGF2, HGF and VEGF; gelatin sponge for hemostasis
associated with transplantation (product name: Spongel; Astellas
Pharma); and tissue adhesives used to fix transplanted tissues,
such as Bolheal (Teijin Pharma), Beriplast.TM. (CSL Behring) and
TachoComb.TM. (CSL Behring).
[0105] The present invention also provides a method of preparing a
non-human chimeric animal, comprising transplanting a non-human
animal with a biological tissue that has been integrated with a
vascular system by coculturing with vascular cells and mesenchymal
cells, and differentiating the biological tissue into a tissue or
an organ in which vascular networks have been constructed. The
non-human animal (such as mouse) transplanted with the biological
tissue integrated with a vascular system can mimic the
physiological function of the animal species (such as human) from
which the vascularized biological tissue is derived. Non-human
animals include, but are not limited to, animals used, for example,
as experimental animals, pet animals, working animals, race horses
or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog,
monkey, cattle, horse, sheep, chicken, shark, devilfish, radish,
salmon, shrimp, crab or the like may be used. Further, in order to
avoid immunorejection, the non-human animal to be used herein is
preferably an immunodeficient animal.
[0106] Further, the present invention also provides a method of
evaluating a drug, comprising using at least one member selected
from the group consisting of the biological tissue integrated with
a vascular system by the above-described method, the tissue or
organ prepared from the vascularized biological tissue, and the
non-human chimeric animal transplanted with the vascularized
biological tissue. Specific examples of drug evaluation include,
but are not limited to, evaluation of drug metabolism (e.g.,
prediction of drug metabolism profiles), evaluation of drug
efficacy (e.g., screening for drugs that are effective as
pharmaceuticals; confirmation of the effect of pharmaceuticals such
as the relationship between drug efficiency and blood vessels;
etc.), toxicity evaluation, and evaluation of drug
interactions.
[0107] With respect to evaluation of drug efficacy, human-type drug
metabolism profiles may be obtained as follows. Briefly, a
biological human tissue integrated with a vascular system, a human
tissue or organ prepared from a biological tissue integrated with a
vascular system, or a non-human chimeric animal transplanted with a
biological human tissue integrated with a vascular tissue is
administered with a candidate compound for pharmaceuticals; then,
biological samples are taken and analyzed. According to these
processes, prediction of the distribution/metabolism/excretion
process of pharmaceuticals in humans--which has been extremely
difficult to achieve by conventional methods--becomes possible and
one may. expect that the development of safe and efficacious
pharmaceuticals can be remarkably accelerated.
[0108] Screening for drugs that are effective as pharmaceuticals is
carried out as follows. Briefly, starting with a tissue induced
from a cell/tissue established from a diseased patient, a
biological tissue integrated with a vascular system, a tissue or an
organ prepared from this vascularized biological tissue, or a
non-human chimeric animal transplanted with this vascularized
biological tissue is prepared. Then, a candidate compound for
pharmaceuticals is administered for analyses. As a result, one may
expect that the prediction accuracy of drug efficacy in actual
administration to humans--which has been insufficient in
conventional in vitro tests--can be greatly improved.
[0109] Confirmation of the relationship between drug efficacy and
blood vessels is achieved as follows. Briefly, a biological tissue
integrated with a vascular system, a tissue or an organ prepared
from this vascularized biological tissue, or a non-human chimeric
animal transplanted with this vascularized biological tissue is
administered with a given drug. Then, the concentration
distribution of the drug in tissues at the vicinity of blood
vessels and the desired drug's effect on cells are measured.
[0110] In tumor tissues, for example, targeting cancer stem cells
which are clinically considered a cause of recurrence or metastasis
is believed to be an important therapeutic strategy. On the other
hand, it is known that when cancer stem cells are present at the
vicinity of blood vessels, vascular permeability is decreased and
anticancer agents are difficult to infiltrate whereas if they are
distant from blood vessels, diffusion of anticancer agents is
insufficient. For developing drugs targeting at cancer stem cells,
it has been important to reconstitute a three-dimensional tumor
tissue that starts from blood vessels and use this tissue for
evaluation. By using the method of the present invention, the
evaluation of drug efficacy based on cell/tissue polarity with
respect to blood vessels which has been entirely unachievable by
conventional methods can be realized and development of drugs with
higher therapeutic effects can be performed.
[0111] In the case of toxicity evaluation, a biological tissue
integrated with a vascular system, a tissue or an organ prepared
from this vascularized biological tissue or a non-human chimeric
animal transplanted with this vascularized biological tissue is
used as a target which is administered a test substance and
thereafter the expressions of tissue disorder markers are measured,
whereby the accuracy in disorder prediction can be improved.
[0112] Development of anticancer agents and other pharmaceuticals
that may have toxicity problems has required huge costs and
prolonged periods for evaluating drug toxicity. By creating a
micro-environment mimicking the inside of a living body using
vascularized tissues, toxicity tests on tissues--which have
heretofore been difficult to evaluate--become available. Briefly,
by carrying out toxicity evaluation on blood vessels, diseased
cells and normal cells, one may expect that the research and
development of new pharmaceuticals can be remarkably expedited.
[0113] Evaluation of drug interactions may be performed as follows.
Briefly, a biological tissue integrated with a vascular system, a
tissue or an organ prepared from this vascularized biological
tissue or a non-human chimeric animal transplanted with this
vascularized biological tissue is used as a target which is
administered with a plurality of drugs; then, examination of each
drug's pharmacokinetics (distribution/metabolism/excretion
processes), toxicity evaluation, and drug efficacy evaluation are
performed.
[0114] The function level of the cells obtained from pluripotent
stem cells by conventional directed differentiation remained less
mature in the differentiation stage than those functional cells
that constitute adult tissues. If, by the method of the present
invention, terminally differentiated functional cells are
obtainable from tissues induced from pluripotent stem cells or the
like, it will be a revolutionary technique of directed
differentiation that serves as an important platform adapted for
industrial production of human functional cells. For example, human
hepatocytes or human hepatic stem cells isolated from the human
liver tissues artificially prepared by the present invention will
enable mass production of human adult hepatocytes which are
necessary for drug discovery and development.
[0115] Further, by integrating cancer tissues or normal tissues
with steric vascular networks, a revolutionary screening technique
will be realized which can evaluate drug efficacy from a totally
new viewpoint such as the correlation between development of drug
efficacy and spatial arrangement of blood vessels--a problem that
has remained unsolved in drug discovery and development.
[0116] Conventionally, medical transplantation targeting such
diseases as diabetes was mainly tissue transplantation therapy
involving the transplantation of islet tissues or the like
extracted from bodies derived from brain-dead donors, for example.
However, engraftment of transplants after the transplantation was
remarkably low because the transplants used in tissue
transplantation therapy had no vascular system. Thus, the
therapeutic effect was rather limited. According to the present
invention, it has become possible to supply vascularized
transplants that can solve this problem. If industrial production
of human tissues/organs for therapeutic purposes that are
integrated with vascular networks becomes possible, new
tissues/organs for transplantation which are expected to provide
higher therapeutic effects can be supplied, potentially serving as
a revolutionary medical technique.
EXAMPLES
[0117] Hereinbelow, the present invention will be described in more
detail with reference to the following Examples.
Example 1
Integration of Vascular Networks for Pancreatic Islet Tissues
[Methods]
1. Isolation of Mouse Pancreatic Islets
[0118] Isolation of mouse pancreatic islets (hereinafter,
frequently referred to simply as "islets") was performed mainly
according to the method of Dong et al. (Title of the document: A
protocol for islet isolation from mouse pancreas). C57BL/6J mice
(Japan SLC, Inc.) anesthetized with diethyl ether (Wako) were
laparotomized after disinfection of the abdomen with 70% ethanol.
The ampulla of Vater (that is a joint between the common bile duct
and the duodenum) was ligated. Subsequently, a 27 G injection
needle was inserted into the site of junction of the cystic duct
and the hepatic duct, and 3 ml of collagenase XI solution (1,000
U/ml) (Sigma, cet. No. C7657) prepared with Hanks' buffer (HBSS,
Gibco) was injected to fill the entire pancreas with collagenase XI
solution. The pancreas was cut out and placed in a 50 ml tube
containing collagenase XI solution, which was then shaken at
37.5.degree. C. for 15 min. After digestion of the pancreas, 25 ml
of ice-cooled HBSS (containing 1 mM CaCl.sub.2) was added to the
tube for washing. Then, the tube was centrifuged (290 g, 30 sec,
4.degree. C.), followed by removal of the supernatant. After
re-washing and re-centrifugation, 15 ml of HBSS was added to the
tube. The resultant content was filtered with a 70 .mu.m mesh cell
strainer. The residue was entirely transferred into a petri dish
using an originally prepared medium [EGM.TM. BulletKit.TM. (Lonza
CC-4133) originally modified for the purpose of culturing
islets].
2. Selection of Mouse Pancreatic Islets
[0119] When the mouse islets isolated in 1 above were observed
under a stereomicroscope, orange-colored spherical mouse islets
(150-250 .mu.m in diameter) could be confirmed. These islets were
transferred to an islet culture medium with a Pipetman.
3. Primary Culture of Mouse Pancreatic Islets
[0120] Mouse islets were cultured using an originally prepared
medium [EGM.TM. BulletKit.TM. (Lonza CC-4133) supplemented with 10%
fetal bovine serum (BWT Lot. S-1560), 20 mmol/L L-glutamine (Gibco)
and 100 .mu.g/ml penicillin/streptomycin (Gibco)] in a 37.degree.
C. 5% CO.sub.2 incubator.
4. Cell Culture
[0121] Normal human umbilical vein endothelial cells (HUVECs)
(Lonza CC-2517) were cultured using a medium prepared especially
for culturing HUVECs [EGM.TM. BulletKit.TM. (Lonza CC-4133)] within
a guaranteed passage number (5 passages). Human mesenchymal stem
cells (hMSCs) (Lonza PT-2501) were cultured using a medium prepared
especially for culturing hMSCs [MSCGM.TM. BulletKit.TM. (Lonza
PT3001)] within a guaranteed passage number (5 passages). Both
HUVECs and hMSCs were cultured in a 37.degree. C., 5% CO.sub.2
incubator.
5. Fluorescence Labeling with Retrovirus Vectors
[0122] All the gene recombination experiments were performed in P2
level safety cabinets under an approval of the Gene Recombination
Committee of Yokohama City University.
[0123] Production of virus vectors pGCD.DELTA.NsamEGFP and
pGCD.DELTA.NsamKO was performed by the method described below.
Briefly, 293GPG/pGCD.DELTA.NsamEGFP cells (kindly provided by Mr.
Masafumi Onodera) and 293GPG/pGCD.DELTA.NsamKO cells (kindly
provided by Mr. Masafumi Onodera) were seeded on
poly-L-lysine-coated dishes and cultured in an especially prepared
medium (designated "293GPG medium"). Briefly, DMEM (Sigma)
containing 10% fetal bovine serum (Gibco), 2 mmol/L L-glutamine
(Gibco), 1.times. penicillin/streptomycin (Gibco), 1 .mu.g/mL
tetracycline hydrochloride (Sigma T-7660), 2 .mu.g/mL puromycin
(Sigma P-7255) and 0.3 mg/mL G418 (Sigma A-1720) was used.
Cultivation was carried out in a 37.degree. C., 10% CO.sub.2
incubator. When cells reached about 80% confluence, the medium was
exchanged with a different medium equivalent to 293GPG medium
except that it was freed of tetracycline hydrochloride, puromycin
and G418 (this medium is designated "293GP medium") (the day of
exchange shall be day 0). After another medium exchange at day 3,
the viruses were recovered together with the medium starting at day
4, followed by filling with 293GP medium again. The recovered
medium was passed through a 0.45 .mu.m filter and stored
temporarily at 4.degree. C. The medium recovered up to day 7 by the
above-described procedures was centrifuged (6000 G, 4.degree. C.,
16 hr). To the resultant pellet, 400 .mu.L of Stempro (Invitrogen)
was added. After shaking at 4.degree. C. for 72 hr, the resultant
solution was recovered and stored at -80.degree. C. (designated
"100-fold concentrated virus solution").
[0124] HUVECs were cultured until they reached 30-50% confluence.
Protamine (Sigma) was added to the medium to give a final
concentration of 0.4 .mu.m/mL To HUVECs, pGCD.DELTA.NsamEGFP was
added. Then, cells were infected in a 37.degree. C., 5% CO.sub.2
incubator for 4 hr and washed with PBS twice. The medium was
exchanged with a fresh one, followed by incubation in a 37.degree.
C., 5% CO.sub.2 incubator again. These operations were repeated
four times and the cells were fluorescence labeled.
6. Examination of Media for Mouse Pancreatic Islets
[0125] Media for pancreatic islets were prepared using RPMI1640
(Wako) and an endothelial cell medium (EGM.TM. BulletKit.TM.)
(Lonza CC-4133) separately. One mouse islet was left standing in
each well of PrimeSurface.TM. 96-well U plates (Sumitomo Bakelite)
filled with respective media, followed by incubation in a
37.degree. C. incubator. Subsequently, 20 .mu.l of LIVE/DEAD.TM.
Cell Imaging Kit (Life Technologies Japan) was added, followed by
incubation in a 37.degree. C., 5% CO.sub.2 incubator for 15 min.
Then, islets were observed under a confocal microscope (LEICA
TCS-SP5).
7. Preparation of Three-Dimensional Tissues with Human Vasculatures
Using 24-Well Flat Bottom Plate
[0126] For the purpose of chronological observation, EGFP-HUVECs
(2.0.times.10.sup.6 cells) and hMSCs (4.0.times.10.sup.5 cells)
were mixed and centrifuged at 950 rpm for 5 min. After removal of
the supernatant, cells were suspended in 20 .mu.l of a medium for
islets, and gel was solidified [Briefly, Matrigel (BD) and the
medium for islets were mixed at 1:1; the resultant solution was
poured into each well (300 .mu.l/well); and the plate was left
standing in a 37.degree. C., 5% CO.sub.2 incubator for 10 min or
more until solidification occurred]. Cells were seeded on each well
of a 24-well flat bottom plate (BD) in which 300 mouse islets/well
had been left standing. After seeding, the plate was left standing
in a 37.degree. C. incubator for 10 min. After 10 minutes, 1 ml of
the medium for islets was added gently down the well wall, followed
by incubation in a 37.degree. C. incubator for one day.
8. Preparation of Three-Dimensional Tissues with Human Vasculatures
Using 96-Well U Plate
[0127] Mouse islets were left standing in each well of
PrimeSurface.TM. 96-Well U Plate (Sumitomo Bakelite) preliminarily
filled with the medium for islets, and HUVECs and hMSCs were seeded
in each well. The plate was subsequently incubated in a 37.degree.
C. incubator for one day.
9. Chronological Observation of Cocultured Cells Using
Stereomicroscope
[0128] Coculture was performed for tracking chronological changes
with a stereomicroscope. Briefly, 10 mouse islets were left
standing in each well of PrimeSurface.TM. 96-Well U Plate. In each
well, HUVECs (1.0.times.10.sup.4 cells) and hMSCs
(1.0.times.10.sup.3 cells) were seeded. After seeding, the plate
was mounted in a stereomicroscope (Leica DFC300FX) to observe
morphological changes caused by coculture.
10. Validation of Islet Cell's Survival Rates Using Transwell
Plate
[0129] Mouse islets (30) were left standing in the bottom of each
well of 24-well Transwell plates. Inserts were placed in other
24-well plates. HUVECs (1.times.10.sup.5 cells), hMSCs
(2.times.10.sup.4 cells) and a mixture of HUVECs (1.times.10.sup.5
cells) and hMSCs (2.times.10.sup.4 cells) were individually seeded
in those inserts, which were then placed in the 24-well plates
where mouse islets had been left standing. The plates were
incubated in a 37.degree. C., 5% CO.sub.2 incubator overnight.
Subsequently, 200 .mu.l of LIVE/DEAD.TM. Cell Imaging Kit (Life
Technologies, Japan) was added to each well of the 24-well plates
where mouse islets had been left standing. Then, the plates were
incubated in a 37.degree. C., 5% CO.sub.2 incubator for 15 min,
followed by observation under a confocal microscope (LEICA
TCS-SP5).
11. Validation of Islet Cell's Survival Rates Using 96-Well U
Plate
[0130] Into the medium for the three-dimensional tissue prepared in
section 8 above, 20 .mu.l of LIVE/DEAD.TM. Cell Imaging Kit (Life
Technologies, Japan) was added, followed by incubation in a
37.degree. C., 5% CO.sub.2 incubator for 15 min. Subsequently,
cells were observed under a confocal microscope.
12. Quantitative Determination of Insulin Secretion Using Transwell
Plate
[0131] Mouse islets (100) were left standing in the bottom of each
well of 24-well Transwell plates. Inserts were placed in other
24-well plates. Inserts in which a mixture of HUVEC
(1.times.10.sup.5 cells) and hMSC (2.times.10.sup.4 cells) was
seeded and inserts in which no cell was seeded were prepared. These
inserts were placed in the 24-well plates where mouse islets had
been left standing. Then, the plates were incubated in a 37.degree.
C., 5% CO.sub.2 incubator overnight. Subsequently, supernatant was
collected from the 24-well plates where mouse islets had been left
standing, and subjected to measurement with an insulin measurement
kit (Shibayagi; Cat. No. AKRIN-011H).
13. Glucose Tolerance Test In Vitro
[0132] Glucose-free RPMI1640 (Wako) was prepared as a medium for
islets. By adding glucose, a low glucose medium (60 mg/100 ml) and
a high glucose medium (360 mg/100 ml) were created. The low glucose
medium was filled in the inserts of 24-well Transwell plate where
mouse islets (100) had been left standing. The inserts were
transferred to wells where a mixture of HUVECs (1.times.10.sup.5
cells) and hMSCs (2.times.10.sup.4 cells) had been seeded, followed
by incubation in a 37.degree. C., 5% CO.sub.2 incubator for 1 hr.
Subsequently, the medium in the inserts was exchanged with the high
glucose medium, and the inserts were transferred to other wells,
followed by incubation in an incubator for 1 hr. After incubation,
supernatants from inserts and wells were collected and subjected to
measurement with an insulin measurement kit (Shibayagi).
14. Experimental Animals
[0133] NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used
as transplantation animal were bred under a SPF environment with a
light-dark cycle consisting of 10 hours for day and 14 hours for
night. The breeding of experimental animals were entrusted to the
Animal Experiment Center, Joint Research Support Section, Advanced
Medical Research Center, Yokohama City University. Animal
experiments were performed in accordance with the ethical
guidelines stipulated by Yokohama City University.
15. Preparation of Cranial Window (CW) Mice for Continuous
Observation
[0134] Preparation of CW mice was performed mainly according to the
method of Yuan et al. (Document Title: Vascular permeability and
microcirculation of gliomas and mammary carcinomas transplanted in
rat and mouse cranial windows). For anesthetization, ketalar
(Sankyo Yell Yakuhin Co., Tokyo, Japan) 90 mg/kg and xylazine
(Sigma Chemical Co., St. Louis, Mo., USA) 9 mg/kg were mixed with
sterilized PBS to give a dose of 200 .mu.l per mouse and
intraperitoneally injected (ketalar/xylazine mixed anesthesia).
Ketalar was used according to the Narcotics Administration Law.
After anesthetization, the hair on the head of NOD/SCID mice was
removed with an electric clipper, and each head was sterilized with
70% ethanol. Then, the skin on the head was incised. The periosteum
on the surface of the skull was removed with cotton swab.
Subsequently, the skull was thinly cut with a dental microdrill
(Fine Science Tools, USA) in a circular manner, and the resultant
circular portion was removed carefully. Then, the dura was scraped
off with tweezers. When bleeding occurred, hemostasis was performed
with spongel (Astellas Co., Tokyo, Japan). After confirmation of
the absence of bleeding, the surface of the brain was filled with
physiological saline (Otsuka Pharmaceutical Co., Tokyo, Japan).
Then, a custom-made circular slide glass 7 mm in diameter
(Matsunami, Osaka, Japan) was mounted on the surface and sealed
tightly with an adhesive prepared by mixing coatley plastic powder
(Yoshida, Tokyo, Japan) with Aron Alpha (Toagosei Co., Tokyo,
Japan) until the mixture became cementitious. One week after the
preparation of CW, those mice which did not have any sign of
bleeding or inflammation at the site of surgery were selected and
used in the subsequent experiments.
16. Preparation of Diabetes Model Mice
[0135] Diabetes model mice were created by administering diphtheria
toxin (DT) to SCID Ins-TRECK-Tg mice (kindly provided by Tokyo
Metropolitan Institute for Clinical Medicine). DT 1 .mu.g/kg was
adjusted with physiological saline to give a dose of 200 .mu.l per
mouse and injected intraperitoneally. After administration, regular
glucose level and body weight were measured every day at 17:00.
Those mice which had a regular glucose level reading of 300 mg/dl
for consecutive three days or more were used as diabetes model
mice. Measurement of glucose levels was performed by Glutest neo
Sensor.TM. (Panasonic, Tokyo) on blood samples taken from the tail
vein.
17. Transplantation into CW Mice
[0136] The CW mice prepared in Section 15 above underwent
transplantation after their brain surfaces were exposed by removing
the glass of the cranial window. Those mice which did not have any
sign of bleeding, inflammation or infection on their brain surfaces
were used. After anesthetization, the area surrounding the cranial
window was disinfected with 70% ethanol. The pointed end of an 18G
needle was inserted into the border line between the custom-made
circular slide glass and Aron Alpha and so manipulated as to peel
off the slide glass without damaging the brain surface. Thus, the
brain surface was exposed. Subsequently, the brain surface was
washed with physiological saline. A tissue transplant was left
standing near the center of the brain surface, and the slide glass
was remounted. To ensure no gap would be left, the space between
the slide glass and the brain surface was filled with physiological
saline and thereafter the slide glass was sealed tightly with an
adhesive prepared from coatley plastic powder and Aron Alpha, in
the same manner as performed at the time of preparation of CW
mouse.
18. Transplantation into the Subcapsular Space of the Kidney
[0137] The diabetes model mice prepared in Section 16 above were
anesthetized with isoflurane using an anesthetizing device for
experimental animals (Shinano). Subsequently, the hair in the left
half of the back of each mouse was removed with an electric
clipper. After the shaven site was disinfected with 70% ethanol,
the kidney was exposed by 1.5-2 cm incision. After exposure, the
kidney was fixed and the capsule on the ventral side of the kidney
was partially incised. Through the resultant opening,
three-dimensional tissues prepared in Section 7 above were
transplanted. After transplantation, the kidney was returned into
the body. Then, the fascia and the skin were sutured.
19. Periodical observation with Confocal Microscope of the Tissues
Transplanted into CW Mice
[0138] The three-dimensional tissues transplanted into CW mice in
Section 17 above were observed.
[0139] Those mice which underwent transplantation were anesthetized
by ketalar/xylazine mixed anesthesia in the same manner as in
Section 11 above. Each mouse was fixed on a 25.times.60 mm micro
cover glass (Matsunami) in the supine position so that the cranial
window would become level. Morphological changes of the
transplanted three-dimensional tissues with vascular networks were
observed with a confocal microscope (LEICA TCS-SP5).
19-1 Visualization of Mouse Blood Flow
[0140] In order to visualize the blood flow from them, the host
mice that underwent transplantation were anesthetized in the same
manner as in Section 15 above. A fluorescent dye prepared by mixing
fluorescein isothio-cyanate-dextran (Sigma, USA) with physiological
saline was administered to each mouse at a rate of 100 .mu.l per 20
g body weight from the tail vein using Myjector 29G. Subsequently,
observation was performed in the same manner as described in
Section 19 above.
19-2 Visualization of Host Derived Vascular Endothelial Cells
[0141] In order to visualize host-derived blood vessels among the
vascular networks constructed in the transplanted cells, mice were
anesthetized in the same manner as in Section 15, followed by
injection of Alexa-Flour 647 anti-mouse CD31 (Biolegend) antibody
at a rate of 100 .mu.l per 20 g body weight from the tail vein
using a 29G syringe. Subsequently, observation was performed in the
same manner as described in Section 19 above.
20. Visualization of Normal Islet Tissues
[0142] The internal structure of normal islet tissues was
visualized using Pdx-DsRed mice (kindly provided by Mr. Douglous
Melton) and CAG-GFP mice (Japan SLC). The mice were anesthetized
with isoflurane using an anesthetizing device for experimental
animals. The hair on the back of each mouse was removed with an
electric clipper. Then, each mouse was incised in the back by 0.5-1
cm so that the spleen was exposed to the outside, whereupon the
pancreas adhering in the vicinity of the spleen became exposed.
After this exposure, each mouse was held in a 10 cm dish such that
the pancreas stuck to the bottom. With each mouse held in this
position, 1.5% agarose gel solution cooled to 37.degree. C. was
poured into the dish to thereby fix the mouse as the pancreas
remained exposed. Normal islet tissues in the fixed mouse were
observed with a confocal microscope.
21. Glucose Tolerance Test In Vivo
[0143] A glucose solution 3 g/kg was adjusted with physiological
saline to give a dose of 200 .mu.l per mouse and administered by
intraperitoneal injection. After administration, blood samples were
taken from the tail vein every 15 min and measured for glucose
levels with a Glutest neo Sensor.TM. (Panasonic, Tokyo).
22. Preparation of Frozen Sections
[0144] Transplanted samples were removed, washed with PBS and fixed
in 4% paraformaldehyde for 1 day. Then, the sample tissue was
transferred into 10% and 20% sucrose solutions, and kept there
until it sank (sucrose replacement). The sinking tissue was
transferred from the 20% sucrose solution to a 30% sucrose solution
and kept there for 1 day for sucrose replacement. The resultant
sample tissue was embedded in O.C.T. compound (Funakoshi Co.),
followed by infiltration at 4.degree. C. for 15 min. Subsequently,
the sample tissue was mounted on a stand of aluminum foil floating
on liquid nitrogen for freezing.
[0145] The resultant frozen block was sliced thinly into 5 .mu.m
thick sections with a cryostat (Lwica CM1950) and adhered onto a
slide glass (Matsunami) Frozen sections were air-dried before
use.
23. Preparation of Paraffin Sections
[0146] Transplanted samples were removed, washed with PBS and fixed
in 4% PFA for 1 day. After fixation, the sample was washed with PBS
three times, and dehydrated with 50, 70, 80, 90, 95 or 100% ethanol
for 1 hr at each concentration. After 1 hr dehydration with 100%
ethanol, the sample was dehydrated with fresh 100% ethanol for 1
day. The resultant sample was subjected to xylene replacement three
times, each for 1 hr and transferred into a thermostat bath for
paraffin embedding that was set at 65.degree. C., where the sample
was infiltrated with a paraffin:xylene (1:1) mixture for 1 hr and
with paraffin three times, each for 2 hr. After infiltration, the
sample was embedded in paraffin to prepare a paraffin block.
[0147] The thus prepared paraffin block was sliced on a microtome
thinly into 5 .mu.m thick sections, which were used as paraffin
sections.
24. HE (Haematoxylin/Eosin) Staining
[0148] Frozen sections were washed with tap water for 2 min to
remove the OCT compound. After washing with deionized water, tissue
sections were nuclear-stained with haematoxylin (Wako) for 9 min.
Subsequently, the stain solution was washed out with deionized
water. The resultant tissue sections were soaked in tap water for
10 min to effect water extraction. Subsequently, after washing with
deionized water, the cytoplasm of tissue sections was stained with
eosin (Muto Chemical) for 10 min. After removing the excessive
eosin with deionized water, tissue sections were dehydrated with a
series of ethanol baths at increasing concentrations, cleared with
xylene, and shielded.
[0149] Paraffin sections were infiltrated with 100% xylene three
times, each for 5 min and then soaked in 100, 90, 80, 70, 60 or 50%
ethanol for 3 min at each concentration to effect deparaffinization
that rendered the sections hydrophilic. Subsequently, similar to
the frozen sections described above, the hydrophilic sections were
washed with deionized water and, thereafter, HE staining was
performed.
25. Immunohistochemical Staining
[0150] After OCT removal and deparaffinization, tissue sections
were each washed with PBS three times for 5 min and fixed in 4% PFA
for 10 min at 4.degree. C. Subsequently, the tissue sections were
washed with PBS three times for 5 min, and blocked at 4.degree. C.
overnight with a blocking solution containing 10% normal serum of
an animal used for secondary antibody preparation (goat). Then, a
primary antibody diluted 200-fold with PBS was added and after
reaction at 4.degree. C. overnight, the sections were washed with
PBS three times for 5 min. As the primary monoclonal antibody, a
combination of anti-mouse/guinea pig insulin antibody,
anti-human/mouse CD31, anti-mouse/rat CD31, anti-human/mouse
collagen 4, anti-human/rabbit laminin antibody, and
anti-mouse/rabbit caspase-3 antibody was used. Further, a secondary
antibody diluted 500-fold with PBS was added to the tissue sections
and after reaction at room temperature under shading conditions for
1 hr, the tissue sections were washed with PBS three times for 5
min, shielded with a mounting medium containing
4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen),
and observed and photographed with a fluorescence microscope. As
the secondary antibody (Molecular Probe), a combination of the
following antibodies was used: Alexa 488-, 555-labeled goat
anti-rabbit IgG.sub.(H+L) antibody, Alexa 488-, 555-, 647-labeled
goat anti-rat IgG.sub.(H+L) antibody, Alexa 488-, 555-labeled goat
anti-guinea pig IgG.sub.(H+L) antibody, and Alexa 488-, 555-,
647-labeled goat anti-mouse IgG.sub.(H+L) antibody.
26. Immunohistological Analysis by Whole Mount Method
[0151] Vascularized islets as generated were recovered and fixed in
a 4% PFA solution for 1 day, followed by washing with PBS three
times for 10 min. After fixation, the islets were placed in a 0.1%
Triton-PBS solution containing 3% BSA and blocked at room
temperature for 1 hr. After blocking, the islets were washed with a
0.1% Triton-PBS solution three times for 10 min. A transplant was
placed in a solution of primary antibody diluted with a 0.1%
Triton-PBS solution and reaction was performed at 4.degree. C. for
1 day. After the reaction, the transplant was washed with a 0.1%
Triton-PBS solution three times for 10 min and then placed in a
solution of secondary antibody diluted with a 0.1% Triton-PBS
solution, followed by reaction at room temperature for 4 hr. After
the reaction, the transplant was washed with a 0.1% Triton-PBS
solution three times for 10 min. A mounting medium containing DAPI
was added to the transplant, which was then observed with a
confocal microscope.
[Results]
1. Generation of Three-Dimensional Tissues by Coculturing Mouse
Islets, Vascular Endothelial Cells and Mesenchymal Stem Cells
[0152] Media were validated using the survival rate of islet cells
as an indicator (FIG. 1A). At 72 hours of culture, dead cell
numbers per islet area under respective conditions were 14
cells/mm.sup.2 in RPMI1640 medium; 1.8 cells/mm.sup.2 in the mixed
medium of RPMI1640 and the endothelial cell medium; and 0.8
cells/mm.sup.2 in the endothelial cell medium (FIG. 1B).
[0153] Culture was performed as described in Section 7 of Methods
above. Immediately after the beginning of culture, cells were
scattered around islets and no three-dimensional tissues visible
with eyes were recognized. At 4 hours of culture, however,
interactions between cells started, and scattered cells began to
gather closely. At 8 hours of culture, cells so aggregated as to
cover islets and gradually constituted a three-dimensional
structure. Finally, at 24 hours of culture, self-organization
progressed further and a vascularized three-dimensional tissue was
constituted (FIG. 1C, upper panel; FIG. 1E). On the other hand,
when coculture was not performed but islets alone were cultured,
neither vascularization nor formation of three-dimensional tissues
was recognized (FIG. 1D).
[0154] Further, by culturing islets as described in Section 8 of
Methods above, an attempt was made to decrease the size of
vascularized three-dimensional tissues in a culture plate
(substrate?) of such a shape that cells/tissues would gather in the
bottom (FIG. 2). When 1, 5, 10 and 20 mouse islet tissues were
cocultured with HUVECs and MSCs, three-dimensional tissues were
formed at 24 hours of culture and their morphology was retained
even at 48 hours of culture (FIG. 2A). Further, minimum cell
numbers of HUVECs and MSCs required for constitution of a
vascularized three-dimensional tissue were examined (FIG. 2B). When
10 mouse islets were cocultured with 1.0.times.10.sup.4 HUVECs and
1.0.times.10.sup.3 MSCs, scattered cells began to aggregate due to
the intercellular adhesion at 2 hours of culture. At 9 hours of
culture in an advanced stage, cells so aggregated as to cover
islets until they constituted a three-dimensional tissue (FIG. 2C,
left panel). In order to track morphological changes in cells,
coculture experiments were performed using fluorescence-labeled
mouse islets and various kinds of cells (FIG. 1A, lower panel; FIG.
2C, right panel; FIG. 2D). Briefly, islets isolated from Pdx-DsRed
mice (FIG. 1A; 2D: red; 2C: blue), HUVECs into which green
fluorescent protein (GFP) had been introduced (FIG. 1A, 2C, 2D:
green) and MSC (FIG. 2C: red) were cocultured, followed by
observation of cell morphology under a confocal microscope.
Immediately after the beginning of culture, HUVECs were found to be
scattered evenly around islets. Further, HUVECs were shown not only
to adhere directly to islet tissues; some of them were also shown
to connect to vascular endothelial cells inside the islets (FIG.
2E).
[0155] From the foregoing, it was revealed that a vascularized
three-dimensional tissue was autonomously generated by coculturing
the three types of cells, i.e., mouse islet, HUVEC and MSC, under
appropriate conditions.
2. Improvement of the Function of Mouse Islet by Coculture with
Vascular Endothelial Cells and Mesenchymal Stem Cells
[0156] Mouse islets were cultured as described in Section 10 of
Methods above and their survival rates under various conditions
were compared (FIG. 1F, viable cell: green; dead cell: red). At 24
hours of culture, dead cell numbers per islet area under the
respective conditions were 53 cells/mm.sup.2 in monoculture of
islets alone, 14 cells/mm.sup.2 in coculture with HUVECs, 2
cells/mm.sup.2 in coculture with MSCs, and 0.1 cells/mm.sup.2 in
coculture with HUVECs and MSCs (FIG. 1G). From these results, it
was shown that the survival rate of mouse islet cells was improved
by coculturing with HUVECs and MSCs.
[0157] Further, culture was performed as described in Section 12 of
Methods above and insulin levels secreted from the mouse islets
were measured (FIG. 1H). At 24 hours of culture, the insulin
secretion from the mouse islets cocultured with HUVECs and MSCs was
greater than that from the monocultured mouse islets. When a
glucose tolerance test was performed in vitro, insulin secretion
increased 1.37-fold in the islet monoculture group and 1.97-fold in
the coculture group (FIG. 1I). In order to specify the group of
molecules contributing to such improvement of islet function,
changes in gene expressions before and after coculture with HUVCs
and MSCs were analyzed comprehensively by microarray analysis. As a
result, 214 candidate genes were extracted as genes whose
expression was enhanced by coculture by a factor of two or more
(FIG. 1J). It was therefore suggested that coculturing mouse islets
with HUVECs and MSCs initiated changes in the expression of various
genes, leading to an improvement of the function of the mouse
islets.
3. Periodical Observation of Vascularized Islet Transplantation
[0158] The vascularized islet generated in Section 1 of Results
above was transplanted into mice and morphological changes in
tissues were tracked (FIG. 3). Further, in order to examine the
necessity of vascularization for generating tissues, mouse islets
alone were transplanted into mice for comparison. Vascularized
islets were transplanted into cranial window (CW) mice as described
in Section 17 of Methods, and morphological changes were tracked as
described in Section 19 of Methods.
[0159] After transplantation of mouse islets alone, no macroscopic
changes were observed in mouse heads until day 2
post-transplantation. Also, no blood perfusion into transplanted
islets was observed. As time passed after transplantation, viable
islets decreased (FIG. 3B). When fluorescence labeling was used to
observe changes in cell morphology, there were no changes, either,
but the number of islets gradually decreased. Further, when blood
flow was visualized, no blood perfusion into the inside of islets
occurred at day 7 post-transplantation (FIG. 3D, islet: green;
blood flow: red). However, in the mouse heads transplanted with
vascularized islets, blood perfusion to all over the
transplantation site occurred at day 2 post-transplantation (FIG.
3A). Further, according to an observation with a confocal
microscope, blood perfusion into the inside of islets was confirmed
at day 7 post-transplantation (FIG. 3C, islet: green; blood flow:
red).
[0160] It was shown by these results that transplantation of
vascularized islets induced early resumption of blood flow into the
inside of the transplanted islets and improved the islet survival
rate after transplantation.
4. Validation of Therapeutic Effect on Diabetes by Transplantation
of Vascularized Islets
[0161] Forty vascularized islets cocultured under the condition of
5 islets were transplanted into the subcapsular space of the kidney
of diabetes model mice and evaluated for their therapeutic effects
(FIG. 3E). Decrease in glucose level was seen at day 1
post-transplantation, and normal glucose level was kept stably
retained at week 2 post-transplantation and thereafter (FIG. 3F).
Further, a great increase in body weight was seen (FIG. 3G) and
survival rate improved (FIG. 3H). The results of a glucose
tolerance test in vivo revealed that the diabetes model mice showed
a insulin secretion response which was almost equal to that of
normal mice (FIG. 3I).
[0162] As described above, therapeutic effects on diabetes were
shown by transplanting vascularized islets.
5. Histological Analysis of Vascularized Islets
[0163] Vascularized islets at day 1 of coculture were analyzed
histologically and immunohistologically. When HE staining was
performed, islet tissues were observed that had no central necrosis
and which adjoined HUVECs and MSCs (FIG. 2E, upper panel). Further,
immunostaining was performed as follows (FIG. 1E'; 2E, lower
panel). Briefly, islets were stained with insulin antibodies (FIG.
1E': green; 2E: red); HUVECs were stained with human vascular
endothelial cell antibodies (FIG. 1E': red; 2E: green); and mouse
blood vessels were stained with mouse vascular endothelial cell
antibodies (FIG. 2E: blue). The presence of HUVECs was confirmed in
the inside of insulin-positive islets, and HUVECs and mouse blood
vessels were connected together.
[0164] Further, vascularized islets (FIG. 3J) and islets (FIG. 3K)
at day 30 post-transplantation into cranial windows were
individually analyzed histologically and immunohistologically. As a
result of HE staining, islets engrafting onto the brain tissue were
confirmed. As a result of immunostaining, it was found that human
vascular endothelial cells were present at insulin-positive sites
in the vascularized islets, and that such human vascular
endothelial cells were stable human blood vessels that would
secrete laminin and collagen IV (extracellular matrices). However,
when islets alone were transplanted, no vascular endothelial cells
were found inside the islets.
[0165] Further, vascularized islets (FIG. 3L) and islets (FIG. 3M)
at day 28 post-transplantation into the subcapsular space of the
kidney were individually analyzed histologically and
immunohistologically. As a result of HE staining, islets present
between the renal parenchyma and the capsule were confirmed (FIG.
3L, lower left panel; FIG. 3M, lower left panel). Further,
immunostaining was performed to stain islets (green) with an
insulin antibody and vascular endothelial cells (red) with a
laminin antibody (FIG. 3L, lower right panel; FIG. 3M, lower right
panel). In the vascularized inlets, expression of laminin-positive
vascular endothelial cells was confirmed inside insulin-positive
islets. However, in those islets which were transplanted with
inlets alone, no vascular endothelial cells were observed.
[0166] As described above, it was shown from histological and
immunohistological viewpoints that the vascularized islets were
islet tissues associated with human blood vessels.
Example 2
Integration of Vascular Networks for Renal Glomeruli
[Methods]
1. Isolation of Mouse Glomeruli
[0167] C57BL/6-Tg mice (Japan SLC, Inc.) anesthetized with diethyl
ether (Wako) were laparotomized after disinfection of the abdomen
with 70% ethanol. The kidney was cut out and the capsule was
removed therefrom. After washing with physiological saline, the
kidney was cut in round slices with a scalpel. The renal pelvis and
the medulla were removed with scissors, and the cortex was
recovered. The recovered cortex was minced on ice and filtered with
a 100 .mu.m mesh cell strainer while adding Hanks' buffer (HBSS,
Gibco) containing 0.1% albumin from bovine serum (BSA, Sigma)
little by little. The flow-through was filtered with a 70 .mu.m
mesh cell strainer, and finally the flow-through was filtered with
a 40 .mu.m mesh cell strainer. The cell mass retained on the 40
.mu.m mesh cell strainer was recovered with 0.1% BSA-containing
Hanks' buffer. The thus recovered material was filtered with a 100
.mu.m mesh cell strainer.
2. Selection of Mouse Glomeruli
[0168] When the mouse glomeruli isolated in Section 1 of Methods
above were observed under a stereomicroscope, spherical mouse
glomeruli (diameter: 50-100 .mu.m) could be confirmed. These
glomeruli were recovered and transferred to a medium for glomeruli
with a Pipetman.
3. Primary Culture of Mouse Glomeruli
[0169] Mouse glomeruli were cultured using RPMI1640 (Wako)
supplemented with 20% fetal bovine serum (BWT Lot. S-1560), 100
.mu.g/ml penicillin/streptomycin (Gibco) and
Insulin-Transferrin-SeleniumX (Gibco) in a 37.degree. C., 5%
CO.sub.2 incubator.
4. Cell Culture
[0170] Normal human umbilical vein endothelial cells (HUVECs)
(Lonza CC-2517) were cultured using a medium prepared especially
for culturing HUVEC [EGM.TM. BulletKit.TM. (Lonza CC-4133)] within
a guaranteed passage number (5 passages). Human mesenchymal stem
cells (hMSCs) (Lonza PT-2501) were cultured using a medium prepared
especially for culturing hMSCs [MSCGM.TM. BulletKit.TM. (Lonza
PT3001)] within a guaranteed passage number (5 passages). Both
HUVECs and hMSCs were cultured in a 37.degree. C., 5% CO.sub.2
incubator.
5. Preparation of Three-Dimensional Tissues Having a Vascular
System
[0171] For the purpose of chronological observation, 1, 5 and 10
mouse glomeruli/well were left standing in each well of
PrimeSurface.TM. 96-Well U Plate (Sumitomo Bakelite) preliminarily
filled with a medium for glomeruli, and 5.times.10.sup.4 HUVECs and
5.times.10.sup.3 hMSCs were seeded in each well. Subsequently, the
plate was incubated in a 37.degree. C. incubator for one day.
Further, 100 mouse glomeruli/well were left standing in each well
of a 24-well plate, and 2.times.10.sup.6 HUVECs and
2.times.10.sup.5 hMSCs were seeded in each well.
6. Chronological Observation Using Stereomicroscope
[0172] Coculture was performed for tracking chronological changes
with a stereomicroscope. Briefly, 20 mouse glomeruli/well were left
standing in each well of a 24-well plate, and 2.times.10.sup.6
HUVECs and 2.times.10.sup.5 hMSCs were seeded in each well. After
seeding, the plate was set in a stereomicroscope (Leica DFC300FX)
and morphological changes caused by coculture were observed.
7. Experimental Animals
[0173] NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used
as transplantation animal were bred under a SPF environment with a
light-dark cycle consisting of 10 hours for day and 14 hours for
night. The breeding of experimental animals were entrusted to the
Animal Experiment Center, Joint Research Support Section, Advanced
Medical Research Center, Yokohama City University. Animal
experiments were performed in accordance with the ethical
guidelines stipulated by Yokohama City University.
8. Transplantation into CW Mice
[0174] The CW mice prepared underwent transplantation after their
brain surfaces were exposed by removing the glass of the cranial
window. Those mice which did not have any sign of bleeding,
inflammation or infection on their brain surfaces were used. After
anesthetization, the area surrounding the cranial window was
disinfected with 70% ethanol. The pointed end of an 18G needle was
inserted into the border between the custom-made circular slide
glass and Aron Alpha and so manipulated to peel off the slide glass
without damaging the brain surface. Thus, the brain surface was
exposed. Subsequently, the brain surface was washed with
physiological saline. A tissue transplant was left standing near
the center of the brain surface, and the custom-made slide glass
was remounted. To ensure no gap would be left, the space between
the slide glass and the brain surface was filled with physiological
saline and thereafter the slide glass was sealed tightly with an
adhesive prepared from coatley plastic powder and Aron Alpha, in
the same manner as performed at the time of preparation of CW
mice.
9. Periodical observation_with Confocal Microscope of the Tissues
Transplanted into CW Mice
[0175] The three-dimensional tissues transplanted into the CW mice
in the preceding Section 8 were observed.
[0176] Those mice which underwent transplantation were anesthetized
by ketalar/xylazine mixed anesthesia in the same manner as in
Section 11 above. Each mouse was fixed on a 25.times.60 mm micro
cover glass (Matsunami) in the supine position so that the cranial
window would become level. Morphological changes of the
transplanted three-dimensional tissues with vascular networks were
observed with a confocal microscope (LEICA TCS-SP5).
[Results]
1. Generation of Vascularized Three-Dimensional Tissues by
Coculture of Mouse Glomeruli, Vascular Endothelial Cells and
Mesenchymal Stem Cells
[0177] Culture was performed as described in Section 6 of Methods
above. Immediately after the beginning of culture, cells were
scattered around glomeruli and no three-dimensional tissues visible
with eyes were recognized. At 4 hours of culture, however,
interactions between cells started, and scattered cells began to
gather closely. At 8 hours of culture in an advanced stage, cells
so aggregated as to cover glomeruli and gradually constituted a
three-dimensional structure. Finally, at 24 hours of culture,
self-organization progressed further and a vascularized
three-dimensional tissue was constituted (FIG. 4A, 4B). On the
other hand, when coculture was not performed but glomeruli alone
were cultured, neither vascularization nor formation of
three-dimensional tissues was recognized (FIG. 4C).
[0178] Further, by culturing glomeruli as described in Section 5 of
Methods above, an attempt was made to decrease the size of
vascularized three-dimensional tissues in a culture plate
(substrate?) of such a shape that cells/tissues would gather in the
bottom (FIG. 4C). When 5, 10 and 15 mouse glomeruli were
individually cocultured with HUVECs and MSCs, three-dimensional
tissues were formed at 24 hours of culture. In order to track
morphological changes in cells, fluorescence-labeled mouse
glomeruli were cocultured with various kinds of cells (FIGS. 4B, 4C
and 4D). Briefly, glomeruli isolated from mice (green), HUVECs into
which Kusabira Orange had been introduced (FIGS. 4B, 4C and 4D:
red) and MSCs (FIGS. 4B and 4D: blue) were cocultured, and cell
morphology was observed with a confocal microscope. It was observed
that, immediately after the beginning of culture, HUVECs were found
to be scattered evenly around glomeruli.
[0179] From the foregoing, it was revealed that a vascularized
three-dimensional tissue was autonomously generated by coculturing
the three types of cells, i.e. mouse glomeruli, HUVEC and MSC,
under appropriate conditions.
2. Periodical Observation of Vascularized Glomeruli
Transplantation
[0180] The vascularized glomeruli generated in Section 1 of Results
above were transplanted into mice and morphological changes in
tissues were tracked (FIG. 4E). Vascularized glomeruli were
transplanted into cranial window (CW) mice as described in Section
8 of Methods, and morphological changes were tracked as described
in Section 9 of Methods.
[0181] In the mouse heads transplanted with the vascularized
glomeruli, blood perfusion to all over the transplantation site
occurred at day 3 post-transplantation (FIG. 4E). Further, the
results of live observation with a confocal microscope at day 10
post-transplantation not only revealed that the glomerular
structure was retained even after transplantation; it was also
found that mouse blood vessels inside glomeruli were directly
anastomosed to human blood vessels (HUVECs), letting blood flow
inside the glomeruli (FIG. 4F). These results show that
transplantation of vascularized glomeruli induced early resumption
of blood flow into the glomeruli and enabled efficient
engraftment.
Example 3
Integration of Vascular Networks for Tumor Tissues
[Methods]
1. Recovery of Human Pancreatic Tumor Tissues
[0182] Human pancreatic tumor tissues removed from nesidioblastosis
patients were washed with PBS under a clean bench environment,
transferred to a 6 cm dish containing a HBSS medium and sliced into
1 mm-square sections, which were used in the subsequent
experiments.
2. Integration of Vascular Networks for Human Pancreatic Tumor
Tissues
[0183] Human pancreatic tumor tissues sliced into 1 mm-square
sections were recovered with a Pipetman (20 sections) and mixed
with 2.times.10.sup.6 EGFP-HUVECs and 2.times.10.sup.5 MSCs. The
mixture was centrifuged at 950 rpm. The resultant supernatant was
removed, and the cells were suspended in 1 ml of EGM medium and
seeded on 24-well plate in which Matrigel was placed in advance.
Then, morphological changes were tracked with a confocal
microscope.
3. Recovery of Mouse Pancreatic Cancer Tissues
[0184] Pancreatic cancer tissues were recovered from pancreatic
cancer model mice (Pdx1-cre; LSL-Kras.sup.G12D; CDKN2A.sup.-/-:
purchased from NCI) which are held to be capable of recapitulating
the multistep carcinogenesis of pancreatic cancer. The cancer
tissues were washed with PBS and transferred to a 6 cm dish
containing a HBSS medium under a clean bench environment. The
recovered cancer tissues were chopped into 1 mm-square sections,
which were used in the subsequent experiments.
4. Integration of Vascular Networks for Mouse Pancreatic Cancer
Tissues
[0185] Pancreatic cancer tissues chopped into 1 mm-square sections
were recovered with a Pipetman (20 sections) and mixed with
2.times.10.sup.6 EGFP-HUVECs and 2.times.10.sup.5 MSCs. The mixture
was centrifuged at 950 rpm for 5 min. The resultant supernatant was
removed, and the cells were suspended in 1 ml of EGM.TM.
BulletKit.TM. (Lonza CC4133) medium and seeded on 24-well plate in
which Matrigel was placed in advance. The plate was incubated in a
37.degree. C. incubator for 4 days while exchanging the medium
every day.
[0186] The 24-well plate was prepared as follows. Briefly, 300
.mu.l of a solution prepared by mixing EGM medium and BD
Matrigel.TM. basement membrane matrix (BD Japan 356234) at 1:1 was
added to each well of a 24-well plate, which was then incubated in
a 37.degree. C. incubator for 10 min to solidify the gel.
[Results]
1. Vascularization of Human Pancreatic Tumor Tissues
[0187] The results of observation with a confocal microscope
confirmed that by means of coculture, vascularized
three-dimensional tissues were autonomously generated in about
24-48 hours while vascular networks were constituted around the
human pancreatic tissues chopped into 1 mm-square sections (FIG.
5A).
2. Vascularization of Mouse Pancreatic Cancer Tissues
[0188] When the pancreatic cancer tissue chopped into 1 mm-square
sections was cocultured with HUVECs and MSCs on the Matrigel.TM.
solidified in 24-well plate, vascularized three-dimensional tissues
could successfully be generated (FIG. 5B, upper panel). As a
control experiment, 1 mm-square sections of the pancreatic cancer
tissue alone were cultured on solidified Matrigel.TM.; neither
formation of three-dimensional tissues nor vascularization was
confirmed and there occurred no changes worth particular mention
(FIG. 5B, lower panel).
[0189] At 4 days of culture, gene expressions in the vascularized
three-dimensional tissues formed were analyzed by quantitative PCR.
The result revealed that the expression of CD44 gene known as an
important cancer stem cell marker increased to a level about 1.6
times as high as the level of expression in the monoculture group
(FIG. 5C).
[0190] It was therefore suggested that cancer stem cells--which
were conventionally difficult to maintain in vitro--were amplified.
Conventional two-dimensional culture systems were difficult to use
as a system for pre-evaluating the efficacy of anticancer agents
because the two-dimensional system has such an environment that the
reactivity of anticancer agents differs greatly from the case where
they are administered in vivo. By using the method of the present
invention, it is expected to reproduce the reactivity in cancer
tissues (including vascular systems) in living bodies. This is a
culture technique that is potentially highly useful as a drug
screening system applicable to the development of novel anticancer
agents.
Example 4
Integration of Vascular Networks for Liver Tissues
[Methods]
1. Isolation of Mouse Liver Tissues
[0191] C57BL/6-Tg mice (Japan SLC, Inc.) anesthetized with diethyl
ether (Wako) were laparotomized after disinfection of the abdomen
with 70% ethanol, followed by transcardial perfusion. The liver was
cut out, washed with physiological saline and minced with scissors.
The minced liver was filtered with a 100 .mu.m mesh cell strainer
while adding Hanks' buffer (HBSS, Gibco) containing 0.1% albumin
from bovine serum (BSA, Sigma) little by little. The flow-through
was filtered with a 70 .mu.m mesh cell strainer. The cell mass
retained on the 70 .mu.m mesh cell strainer was recovered with a
0.1% BSA-containing Hanks' buffer.
2. Primary Culture of Mouse Liver Tissues
[0192] Mouse liver tissues were cultured in DMEM/F12 (Invitrogen)
supplemented with 10% fetal bovine serum (ICN Lot. 7219F), 2 mmol/L
L-glutamine (Gibco), 100 .mu.g/mL penicillin/streptomycin (Gibco),
10 mmol/L nicotinamide (Sigma), 50 .mu.mol/L 2-Mercaptoethanol,
1.times.10.sup.-7 mol/L 6.5% dexamethasone (Sigma),
2.6.times.10.sup.-4 M L-Ascorbic acid 2-phosphate sesquimagnesium
salt hydrate (Sigma), 5 mmol/L HEPES (DOJINDO), 1 .mu.g/mL Human
recombinant insulin expressed in yeast (Wako), 50 ng/mL Human
recombinant HGF expressed in Sf21 insect cells (Sigma) and 20 ng/mL
Mouse Submaxillary Glands EGF (Sigma) in a 37.degree. C., 5%
CO.sub.2 incubator.
3. Cell Culture
[0193] Normal human umbilical vein endothelial cells (HUVECs)
(Lonza CC-2517) were cultured using a medium prepared especially
for culturing HUVECs [EGM.TM. BulletKit.TM. (Lonza CC-4133)] within
a guaranteed passage number (5 passages). Human mesenchymal stem
cells (hMSCs) (Lonza PT-2501) were cultured using a medium prepared
especially for culturing hMSCs [MSCGM.TM. BulletKit.TM. (Lonza
PT3001)] within a guaranteed passage number (5 passages). Both
HUVECs and hMSCs were cultured in a 37.degree. C., 5% CO.sub.2
incubator.
4. Preparation of Three-Dimensional Tissues with Vascular
Networks
[0194] For chronological observation, two mouse liver tissues were
left standing in each well of PrimeSurface.TM. 96-well U plate
(Sumitomo Bakelite) preliminarily filled with a medium for liver
tissues. Then, 5.times.10.sup.4 HUVECs and 5.times.10.sup.3 hMSCs
were seeded in each well. The plate was then incubated in a
37.degree. C. incubator for 1 day.
5. Experimental Animals
[0195] NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used
as transplantation animal were bred under a SPF environment with a
light-dark cycle consisting of 10 hours for day and 14 hours for
night. The breeding of experimental animals were entrusted to the
Animal Experiment Center, Joint Research Support Section, Advanced
Medical Research Center, Yokohama City University. Animal
experiments were performed in accordance with the ethical
guidelines stipulated by Yokohama City University.
6. Transplantation into CW Mice
[0196] The CW mice prepared in Section 8 above underwent
transplantation after their brain surfaces were exposed by removing
the glass of the cranial window. Those mice which did not have any
sign of bleeding, inflammation or infection on their brain surfaces
were used. After anesthetization, the area surrounding the cranial
window was disinfected with 70% ethanol. The pointed end of an 18G
needle was inserted into the border line between the custom-made
circular slide glass and Aron Alpha and so manipulated as to peel
off the slide glass without damaging the brain surface. Thus, the
brain surface was exposed. Subsequently, the brain surface was
washed with physiological saline. A tissue transplant was left
standing near the center of the brain surface, and the slide glass
was remounted. To ensure no gap would be left, the space between
the slide glass and the brain surface was filled with physiological
saline and, thereafter, the slide glass was sealed tightly with an
adhesive prepared from coatley plastic powder and Aron Alpha, in
the same manner as performed at the time of preparation of CW
mouse.
7. Periodical Observation with Confocal Microscope of the Tissues
Transplanted into CW Mice
[0197] The three-dimensional tissues transplanted into CW mice in
Section 6 above were observed.
Those mice which underwent transplantation were anesthetized by
ketalar/xylazine mixed anesthesia in the same manner as in Section
11 above. Each mouse was fixed on a 25.times.60 mm micro cover
glass (Matsunami) in the supine position so that the cranial window
would become level. Morphological changes of the transplanted
three-dimensional tissues with vascular networks were observed with
a confocal microscope (LEICA TCS-SP5).
[Results]
1. Generation of Three-Dimensional Tissues by Coculturing Mouse
Liver Tissues, Vascular Endothelial Cells and Mesenchymal Stem
Cells
[0198] Culture was performed as described in Section 4 of Methods
above. Immediately after the beginning of culture, cells were
scattered around liver tissues, and no three-dimensional tissues
visible with eyes were recognized. At 4 hours of culture, however,
interactions between cells started, and scattered cells began to
gather closely. At 8 hours of culture in an advanced stage, cells
so aggregated as to cover liver tissues and gradually constituted a
three-dimensional structure. Finally, at 24 hours of culture,
self-organization progressed further and a vascularized
three-dimensional tissue was constituted (FIG. 6A, 6B). On the
other hand, when coculture was not performed but liver tissues
alone were cultured, neither vascularization nor formation of
three-dimensional tissues was recognized (FIG. 6B).
[0199] Further, by culturing cells as described in Section 4 of
Methods above, an attempt was made to decrease the size of
vascularized three-dimensional tissues in a culture plate
(substrate?) of such a shape that cells/tissues would gather in the
bottom (FIG. 6A). When mouse liver tissues were cocultured with
HUVECs and MSCs, three-dimensional tissues were formed at 24 hours
of culture. In order to track morphological changes in cells,
coculture experiments were performed using fluorescence-labeled
mouse liver tissues and various kinds of cells (FIG. 6A). Briefly,
liver tissues isolated from mice (FIG. 6A: red; 6B, 6D: green),
HUVECs into which green fluorescent protein (GFP) had been
introduced (FIG. 6B) and MSCs were cocultured, followed by
observation of cell morphology under a confocal microscope.
Immediately after the beginning of culture, HUVECs were confirmed
to be scattered evenly around liver tissues.
[0200] From the foregoing, it was revealed that a vascularized
three-dimensional tissue was autonomously generated by coculturing
the three types of cells, i.e., mouse liver tissue, HUVEC and MSC,
under appropriate conditions.
2. Periodical Observation of Vascularized Liver Tissue
Transplantation
[0201] The vascularized liver tissues generated in Section 1 of
Results above were transplanted into mice, and morphological
changes in tissues were tracked (FIG. 6C). Transplantation into CW
mice was performed as described in Section 6 of Methods, and
morphological changes were tracked as described in Section 7 of
Methods.
[0202] In the heads of mice transplanted with vascularized liver
tissues, blood perfusion to all over the transplantation site
occurred at day 3 post-transplantation (FIG. 6C). Further, when
observed with a confocal microscope, blood perfusion into the
inside of transplanted liver tissues was confirmed (FIG. 6D).
[0203] It was shown by these results that transplantation of
vascularized liver tissues induced early resumption of blood flow
into the inside of transplanted liver tissues.
Example 5
Integration of Vascular Networks for Intestinal Tissues
[Methods]
1. Isolation of Mouse Intestinal Tissues
[0204] C57BL/6-Tg mice (Japan SLC, Inc.) anesthetized with diethyl
ether (Wako) were laparotomized after disinfection of the abdomen
with 70% ethanol. The inlet of the small intestine was cut off by a
length of about 20 cm. The lumen of the small intestine thus cut
off was washed with 50 ml of physiological saline and then cut
lengthwise to expose the mucosa which was cut into small sections
of about 5 cm. Subsequently, the resultant small sections were
treated in PBS containing 2 mM Ethylenediaminetetraacetic acid
(EDTA; Dojinkagaku) and 0.5 mM Dithiothreitol (DTT; Sigma Chemical
Company) at 37.degree. C. for 20 min. The resultant supernatant was
passed through a 100 .mu.m mesh cell strainer and washed with PBS
three times. Finally, the flow-through was filtered with a 40 .mu.m
mesh cell strainer. The cell mass retained on the 40 .mu.m mesh
cell strainer was recovered with a 0.1% BSA-containing Hanks'
buffer.
3. Primary Culture of Mouse Intestinal Tissues
[0205] Mouse intestinal tissues were cultured using RPMI1640 (Wako)
supplemented with 20% fetal bovine serum (BWT Lot. S-1560), 100
.mu.g/ml penicillin/streptomycin (Gibco) and
Insulin-Transferrin-SeleniumX (Gibco) in a 37.degree. C., 5%
CO.sub.2 incubator.
4. Cell Culture
[0206] Normal human umbilical vein endothelial cells (HUVECs)
(Lonza CC-2517) were cultured using a medium prepared especially
for culturing HUVECs [EGM.TM. BulletKit.TM. (Lonza CC-4133)] within
a guaranteed passage number (5 passages). Human mesenchymal stem
cells (hMSCs) (Lonza PT-2501) were cultured using a medium prepared
especially for culturing hMSCs [MSCGM.TM. BulletKit.TM. (Lonza
PT3001)] within a guaranteed passage number (5 passages). Both
HUVECs and hMSCs were cultured in a 37.degree. C., 5% CO.sub.2
incubator.
5. Preparation of Three-Dimensional Tissues with Vascular
Networks
[0207] For chronological observation, 20 mouse intestinal tissues
were left standing in each well of PrimeSurface.TM. 96-well U plate
(Sumitomo Bakelite) preliminarily filled with a medium for
intestinal tissues. Then, 5.times.10.sup.4 HUVECs and
5.times.10.sup.3 hMSCs were seeded in each well. The plate was then
incubated in a 37.degree. C. incubator for 1 day.
6. Chronological Observation of Cell Coculture with
Stereomicroscope
[0208] Coculture was performed for tracking chronological changes
with a stereomicroscope. Briefly, mouse intestinal tissues were
left standing in each well of a 24-well plate, and 2.times.10.sup.6
HUVECs and 2.times.10.sup.5 hMSCs were seeded in each well. After
seeding, the plate was set in a stereomicroscope (Leica DFC300FX)
and morphological changes caused by coculture were observed.
7. Experimental Animals
[0209] NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used
as transplantation animal were bred under a SPF environment with a
light-dark cycle consisting of 10 hours for day and 14 hours for
night. The breeding of experimental animals were entrusted to the
Animal Experiment Center, Joint Research Support Section, Advanced
Medical Research Center, Yokohama City University. Animal
experiments were performed in accordance with the ethical
guidelines stipulated by Yokohama City University.
8. Transplantation into CW Mice
[0210] The CW mice prepared underwent transplantation after their
brain surfaces were exposed by removing the glass of the cranial
window. Those mice which did not have any sign of bleeding,
inflammation or infection on their brain surfaces were used. After
anesthetization, the area surrounding the cranial window was
disinfected with 70% ethanol. The pointed end of an 18G needle was
inserted into the border between the custom-made circular slide
glass and Aron Alpha and so manipulated as to peel off the slide
glass without damaging the brain surface. Thus, the brain surface
was exposed. Subsequently, the brain surface was washed with
physiological saline. A transplant was left standing near the
center of the brain surface, and the custom-made slide glass was
remounted. To ensure no gap would be left, the space between the
slide glass and the brain surface was filled with physiological
saline and, thereafter, the slide glass was sealed tightly with an
adhesive prepared from coatley plastic powder and Aron Alpha, in
the same manner as performed at the time of preparation of CW
mice.
9. Periodical observation_with Confocal Microscope of the Tissues
Transplanted into CW Mice
[0211] The three-dimensional tissues transplanted into CW mice in
the preceding Section 8 were observed.
[0212] Those mice which underwent transplantation were anesthetized
by ketalar/xylazine mixed anesthesia in the same manner as in
Section 11 above. Each mouse was fixed on a 25.times.60 mm micro
cover glass (Matsunami) in the supine position so that the cranial
window would become level. Morphological changes of the
transplanted three-dimensional tissues with vascular networks were
observed with a confocal microscope (LEICA TCS-SP5).
[Results]
1. Generation of Vascularized Three-Dimensional Tissues by
Coculture of Mouse Intestinal Tissues, Vascular Endothelial Cells
and Mesenchymal Stem Cells
[0213] Culture was performed as described in Section 4 of Methods
above Immediately after the beginning of culture, cells were
scattered around intestinal tissues, and no three-dimensional
tissues visible with eyes were recognized. At 4 hours of culture,
however, interactions between cells started, and scattered cells
began to gather closely. At 8 hours of culture in an advanced
stage, cells so aggregated as to cover intestinal tissues and
gradually constituted a three-dimensional structure. Finally, at 24
hours of culture, self-organization progressed further and a
vascularized three-dimensional tissue was constituted (FIG. 7A,
7B). On the other hand, when coculture was not performed but
intestinal tissues alone were cultured, neither vascularization nor
formation of three-dimensional tissues was recognized (FIG.
7B).
[0214] Further, by culturing as described in Section 4 of Methods
above, an attempt was made to decrease the size of vascularized
three-dimensional tissues in a culture plate (substrate?) of such a
shape that cells/tissues would gather in the bottom (FIG. 7B). When
mouse intestinal tissues were cocultured with HUVECs and MSCs,
three-dimensional tissues were formed at 24 hours of culture. In
order to track morphological changes in cells, fluorescence-labeled
mouse intestinal tissues were cocultured with various kinds of
cells (FIG. 7B). Briefly, intestinal tissues isolated from mice
(FIG. 7B: red), HUVECs into which green fluorescent protein (GFP)
had been introduced (FIG. 7B) and MSCs were cocultured, and cell
morphology was observed with a confocal microscope Immediately
after the beginning of culture, HUVECs were confirmed to be
scattered evenly around intestinal tissues.
[0215] From the foregoing, it was revealed that a vascularized
three-dimensional tissue was autonomously generated by coculturing
the three types of cells, i.e., mouse intestinal tissue, HUVEC and
MSC, under appropriate conditions.
2. Periodical Observation of Vascularized Intestinal Tissue
Transplantation
[0216] The vascularized intestinal tissues generated in Section 1
of Results above were transplanted into mice and morphological
changes in tissue were tracked (FIG. 7C). Vascularized intestinal
tissues were transplanted into cranial window (CW) mice as
described in Section 6 of Methods, and morphological changes were
tracked as described in Section 7 of Methods.
[0217] In the mouse heads transplanted with vascularized intestinal
tissues, blood perfusion to all over the transplantation site
occurred at day 3 post-transplantation (FIG. 7C). Further,
observation with a confocal microscope confirmed that blood
perfusion into the inside of the transplanted intestinal tissues
occurred at day 3 post-transplantation. (FIG. 7D).
[0218] It was shown by these results that transplantation of
vascularized intestinal tissues induced early resumption of blood
flow into the inside of the transplanted intestinal tissues.
Example 6
Integration of Vascular Networks for Pulmonary Tissues
[Methods]
1. Isolation of Mouse Pulmonary Tissues
[0219] C57BL/6-Tg mice (Japan SLC, Inc.) anesthetized with diethyl
ether (Wako) were laparotomized after disinfection of the abdomen
with 70% ethanol, and the lungs were cut out. The lungs were washed
with physiological saline and minced with scissors. The minced lung
was filtered with a 100 .mu.m mesh cell strainer while adding
Hanks' buffer (HBSS, Gibco) containing 0.1% albumin from bovine
serum (BSA, Sigma) little by little. The flow-through was filtered
with a 40 .mu.m mesh cell strainer. The cell mass retained on the
40 .mu.m mesh cell strainer was recovered with a 0.1%
BSA-containing Hanks' buffer.
2. Cell Culture
[0220] Normal human umbilical vein endothelial cells (HUVECs)
(Lonza CC-2517) were cultured using a medium prepared especially
for culturing HUVECs [EGM.TM. BulletKit.TM. (Lonza CC-4133)] within
a guaranteed passage number (5 passages). Human mesenchymal stem
cells (hMSCs) (Lonza PT-2501) were cultured using a medium prepared
especially for culturing hMSCs [MSCGM.TM. BulletKit.TM. (Lonza
PT3001)] within a guaranteed passage number (5 passages). Both
HUVECs and hMSCs were cultured in a 37.degree. C., 5% CO.sub.2
incubator.
3. Preparation of Three-Dimensional Tissues with Vascular
Networks
[0221] For chronological observation, 20 mouse pulmonary tissues
were left standing in each well of PrimeSurface.TM. 96-well U plate
(Sumitomo Bakelite) filled with a medium for pulmonary tissues.
Then, 5.times.10.sup.4 HUVECs and 5.times.10.sup.3 hMSCs were
seeded in each well. The plate was then incubated in a 37.degree.
C. incubator for 1 day. Further, mouse pulmonary tissues were left
standing in each well of a 24-well plate. Then, 2.times.10.sup.6
HUVECs and 2.times.10.sup.5 hMSCs were seeded in each well.
4. Chronological Observation of Cell Coculture with
Stereomicroscope
[0222] Coculture was performed for tracking chronological changes
with a stereomicroscope. Briefly, 20 mouse pulmonary tissues were
left standing in each well of a 24-well plate. HUVECs
(2.times.10.sup.6 cells) and hMSCs (2.times.10.sup.5 cells) were
seeded in each well. After seeding, the plate was set in a
stereomicroscope (Leica DFC300FX) and morphological changes caused
by coculture were observed.
5. Experimental Animals
[0223] NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used
as transplantation animal were bred under a SPF environment with a
light-dark cycle consisting of 10 hours for day and 14 hours for
night. The breeding of experimental animals were entrusted to the
Animal Experiment Center, Joint Research Support Section, Advanced
Medical Research Center, Yokohama City University. Animal
experiments were performed in accordance with the ethical
guidelines stipulated by Yokohama City University.
6. Transplantation into CW Mice
[0224] The CW mice prepared in Section 8 underwent transplantation
after their brain surfaces were exposed by removing the glass of
the cranial window. Those mice which did not have any sign of
bleeding, inflammation or infection on their brain surfaces were
used. After anesthetization, the area surrounding the cranial
window was disinfected with 70% ethanol. The pointed end of an 18G
needle was inserted into the border line between the custom-made
circular slide glass and Aron Alpha and so manipulated as to peel
off the slide glass without damaging the brain surface. Thus, the
brain surface was exposed. Subsequently, the brain surface was
washed with physiological saline. A tissue transplant was left
standing near the center of the brain surface, and the slide glass
was remounted. To ensure no gap would be left, the space between
the slide glass and the brain surface was filled with physiological
saline and, thereafter, the slide glass was sealed tightly with an
adhesive prepared from coatley plastic powder and Aron Alpha, in
the same manner as performed at the time of preparation of CW
mouse.
7. Periodical observation with Confocal Microscope of the Tissues
Transplanted into CW Mice
[0225] The three-dimensional tissues transplanted into CW mice in
Section 9 were observed.
[0226] Those mice which underwent transplantation were anesthetized
by ketalar/xylazine mixed anesthesia in the same manner as in
Section 11 above. Each mouse was fixed on a 25.times.60 mm micro
cover glass (Matsunami) in the supine position so that the cranial
window would become level. Morphological changes of the
transplanted three-dimensional tissues with vascular networks were
observed with a confocal microscope (LEICA TCS-SP5).
[Results]
1. Generation of Three-Dimensional Tissues by Coculturing Mouse
Pulmonary Tissues, Vascular Endothelial Cells and Mesenchymal Stem
Cells
[0227] Culture was performed as described in Section 6 of Methods
above. Immediately after the beginning of culture, cells were
scattered around pulmonary tissues, and no three-dimensional
tissues visible with eyes were recognized. At 4 hours of culture,
however, interactions between cells started, and scattered cells
began to gather closely. At 8 hours of culture in an advanced
stage, cells so aggregated as to cover pulmonary tissues and
gradually constituted a three-dimensional structure. Finally, at 24
hours of culture, self-organization progressed further and a
vascularized three-dimensional tissue was constituted (FIG. 8A). On
the other hand, when coculture was not performed but pulmonary
tissues alone were cultured, neither vascularization nor formation
of three-dimensional tissues was recognized (FIG. 8A).
[0228] Further, by culturing cells as described in Section 4 of
Methods above, an attempt was made to decrease the size of
vascularized three-dimensional tissues in a culture plate
(substrate?) of such a shape that cells/tissues would gather in the
bottom (FIG. 2). When mouse pulmonary tissues were cocultured with
HUVEC and MSC, three-dimensional tissues were formed at 24 hours of
culture. In order to track morphological changes in cells,
coculture experiments were performed using fluorescence-labeled
mouse pulmonary tissues and various kinds of cells (FIG. 8A).
Briefly, pulmonary tissues isolated from mice (FIG. 8A: red),
HUVECs into which green fluorescent protein (GFP) had been
introduced (FIG. 8A: green) and MSC were cocultured, followed by
observation of cell morphology under a confocal microscope.
Immediately after the beginning of culture, HUVECs were confirmed
to be scattered evenly around pulmonary tissues.
[0229] From the foregoing, it was revealed that a vascularized
three-dimensional tissue was autonomously generated by coculturing
the three types of cells, i.e., mouse pulmonary tissue, HUVEC and
MSC, under appropriate conditions.
2. Periodical Observation of Vascularized Pulmonary Tissue
Transplantation
[0230] The vascularized pulmonary tissues generated in Section 1 of
Results above were transplanted into mice, and morphological
changes in tissues were tracked (FIG. 8B). Transplantation into CW
mice was performed as described in Section 16 of Methods, and
morphological changes were tracked as described in Section 7 of
Methods.
[0231] In the heads of mice transplanted with vascularized
pulmonary tissues, blood perfusion to all over the transplantation
site occurred at day 3 post-transplantation (FIG. 8B). Further,
when observed with a confocal microscope, blood perfusion into the
inside of transplanted liver tissues was confirmed at day 7
post-transplantation (FIG. 8C).
[0232] It was shown by these results that transplantation of
vascularized pulmonary tissues induced early resumption of blood
flow into the inside of transplanted pulmonary tissues.
Example 7
Integration of Vascular Networks for iPS Cell-Derived Endodermal
Tissues
[Methods and Results]
1. Directed Differentiation of iPS Cells
[0233] Expanded but undifferentiated iPS cells (kindly provided by
Dr. Nakauchi, Tokyo University; TkDA3 clone; established from
dermal fibroblasts) were washed once with a washing medium
(DMEM/F12; Life Technologies 11320). A cultured cell dissociating
solution (Funakoshi AT104) was added to 100 mm dishes in an amount
of 1 ml per dish. Cells were recovered into 50 ml centrifugal tubes
and subjected to centrifugation at 900 rpm for 5 min. After taking
a cell count, cells were seeded on Matrigel.TM.-coated 60 mm dishes
at a density of 1.5.times.10.sup.6 cells per dish.
Matrigel.TM.-coating was performed as follows. Briefly, BD
Matrigel.TM. basement membrane matrix (BD Japan 356231) was diluted
30-fold with DMEM (Life Technologies 1196118). The thus diluted gel
was added to 60 mm dishes (2 ml/dish), which were left standing at
room temperature for 2 hr. As a culture broth, an iPS culture
medium supplemented with ROCK inhibitor Y-27632 (Calbiochem 688000)
was used. Cells were incubated in a 37.degree. C. incubator for 24
hr to induce cell adhesion. Subsequently, the culture broth was
exchanged with a directed differentiation medium. This medium was
RPMI-1640 (Wako Pure Chemicals 189-02025) supplemented with
B-27.TM. Supplement Minus Insulin (Life Technologies 0050129SA)
(1/100 dilution) and 100 ng/.mu.l Activin A (Ajinomoto). While
exchanging the medium every 2 days, cells were cultured for 6 days
to allow directed differentiation into definitive endoderms. The
degree of differentiation into endodermal lineage was confirmed by
quantitative PCR and immunostaining.
2. Preparation of iPS Cell-Derived Endodermal Tissues
[0234] Human iPS cells which had undergone directed differentiation
into definitive endoderms were seeded in each well of EZSPHERE.TM.
(Asahi Glass 4810-900 6-well-Flat bottom) at a density of
1.0.times.10.sup.6 cells/well. As a culture broth, a 1:1 mixture of
a medium kit for sole use with hepatocytes (HCM.TM. BulletKit.TM.;
Lonza CC3198) and EGM.TM. BulletKit.TM. (Lonza CC-4133) was used.
Cells were cultured in a 37.degree. C. incubator for 8 days, with
half of the medium exchanged every 2 days, to thereby prepare
steric endodermal tissues of 50-500 .mu.m in diameter.
3. Preparation of Three-Dimensional Tissues with Human Vasculatures
Using 96-Well U Plate
[0235] One to twenty iPS cell-derived endodermal tissues were left
standing in each well of PrimeSurface.TM. 96-Well U Plate (Sumitomo
Bakelite) preliminarily filled with the medium for culturing iPS
cell-derived endodermal tissues described in Section 2 above. Then,
1.0.times.10.sup.4 HUVECs and 1.0.times.10.sup.3 hMSCs were seeded
in each well. Subsequently, the cells were incubated in a
37.degree. C. incubator for 4 days.
[0236] As a result, it became clear that endodermal tissues, when
cocultured with human vascular endothelial cells and mesenchymal
stem cells, autonomously induced a three-dimensional tissue (FIG.
9B). It was also found that in the thus induced tissue, human
vascular endothelial cells formed lumen-like structures to form a
vascularized tissue (FIG. 9C). Since the formation of such a
three-dimensional tissue was never confirmed in the monoculture
group of iPS cell-derived endodermal tissues, it was demonstrated
that use of the method of the present invention is essential for
preparing vascularized tissues (FIG. 9B).
[0237] All publications, patents and patent applications cited
herein are incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
[0238] Biological tissues integrated with a vascular system
according to the present invention are applicable to generation of
human functional cells, organ transplantation, drug discovery
screening, new analytical systems for evaluating such factors as
the relationship between development of drug efficacy and blood
vessels.
* * * * *