Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
7 ~ ~
Technical ~ield
The present invention relates to a cell cluster or
sheet comprising a plurality of cell species and a method
of preparing the same. More specifically, it relates to a
cell cluster or sheet comprising a plurality of cell
species having a regulated size and a regulated composition
of cell species. The cell cluster or sheet is useful for
efficient production of cellular products and also useful
as a prosthesis for repairing damaged or diseased parts of
living tissue or as a simulation system to evaluate the
effect of drugs and so on, on a living body.
Bac~ground Art
Organs or tissues are formed from an assembly of
different species of cells, and specific functions of
organs or tissues are caused and controlled by
interactive behaviors of different species of cells. For
example, blood vessel is made of tunica intima, tunica
media and tunica adventitia. On tunica intima there is a
layer of endothelial cells, in tunica media smooth muscle
cells exist and in tunica adventitia there are
fibroblasts. It is known that various kinds of chemical
mediators and hormones are secreted from each cell and
control the functions of the other cells. For example, a
hormone which targets smooth muscle cell contraction is
secreted from endothelial cells. In addition, a growth
factor secreted from fibroblasts enhances the
proliferation of endothelial cells.
On the other hand, skin is made of epidermis and
dermis, and each layer is made of epithelial cells and
fibroblasts. There are factors which are secreted from
the fibroblasts to control proliferation and
differentiation of epithelial cells and factors which are
~0~37~
secreted from the epithelial cells to enhance
proliferation of fibroblasts. Mutual cell interaction is
known to effectively control the functions of skin.
Liver is made up of parenchymal cells, hepatocytes
and sinusoidal cells including endothelial cells,
stellate cells and so on. These cells are believed to
control liver function. Accordingly, a co-culture system
using different cell species which form the tissue or
organ is considered to be much more adequate for
maintaining the tissue or organ specific functions than
mono-culture systems made from only one cell species.
The following evidence indicates the performance of
co-culture systems. A co-culture system comprising islet
cells and fibroblasts significantly increased insulin
secretion from the islet cells as compared with a
mono-culture system of islet cells alone (Rabionovitch,
A. et al., DIABE~ES (1979) 28:1108). A co-culture system
comprising hepatocytes and fibroblasts effectively
prolonged the viability of the hepatocytes as compared
2~ with a mono-culture system of hepatocytes alone
(Yoshizato, K., Abstract of the 4th Meeting for Primary
Culture of Hepatocytes (1988) 11).
Most conventional co-culture systems have been
carried out by culturing a mixture of different cell
species or sequentially culturing different cell species
on culture substrates. Namely, the co-culture systems
developed so far are two-dimensional. Two-dimensional
culture is effective for cell proliferation processes but
inadequate for cell differentiation. Since cell
differentiation is necessary for most cells to exhibit in
vivo specific functions, co-culture of cells on a two-
dimensional substrate is not optimal. The problems
associated with two-dimensional co-culture systems exist
8 ~
-- 4 --
because most in vivo cells are not present in a
two-dimensional mode but are in a three-dimensional mode.
Further, the two-dimensional cu:Lture substrate binds and
restricts the cells so strongly that the cells find it
difficult to regenerate in vivo specific structures and
to exhibit in vivo functions.
An additional problem associated with conventional
two-aimensional substrate co-culture is that it is almost
impossible to harvest the cultured cells from the
substrate and maintain a usable shape, such as a cell
sheet or a cell cluster. Difficulties associated with
conventional harvesting are due to the use of cell
detaching agents, such as trypsin and EDTA. The cell
detaching agents destroy cell-to-cell junctions which are
necessary to maintain the shape such as a cell sheet and
a cell cluster. It is necessary to maintain the cell
cluster or sheet shape in order to use the co-cultured
cells as a prosthesis to repair damaged or diseased
focuses and/or simulate systems of tissues or organs to
evaluate biological activity of test substances.
Attempts have been made to prepare a cell sheet or
cell cluster without the use of cell detaching agents.
Conventional attempts can be classified into two types.
The first method makes cell clusters by spontaneously
aggregating suspended cells on non-adhesive substrates
such as agar and agarose coating (Carlsson, J., et al.,
Spheroids in Cancer Research ( 1984), 1, edited by Acker,
H., et al., Springer-Verlag), and polyhydroxyethyl
methacrylate coating (Landry, J., et al., J. Cell Biol.
(1985) 101:914). The second method makes cell clusters
by spontaneously detaching the cells without use of
detaching agents from semi-adhesive substrates on to
which the cells are weakly attached. Proteoglycans
~ 7
-- 5
coating (Koide, N., et al., ~ioche~. Biophys . Res . Commu .
(1989) 161:385) and positively-charged polystyrene
(Koide, N., et al., Experimenta.Z Cell ~esearch (1990)
186:227) have been used as as such semi-adhesive
substrates. These two methods have been applied to the
co-culture systems of a plurality of cell species. A
cell cluster composed of HeLa cells and fibroblasts was
prep~red by the first method using an agar coated
substrate (Sasaki, T., et al., Cancer Research (1984)
44:345). A cell cluster composed of hepatocytes and
endothelial cells was prepared by the second method using
a proteoglycan coated substrate (Matsushima, K., et al.,
Jpn. ~T, Artif. Organs (1990) 19:848).
The major problems associated with the two
conventional methods for preparing cell clusters or
sheets are the following.
(1) The conventional methods are not applicable to
all cell species. The cell clusters or sheets
formed by these methods are strongly dependent
upon the aggregability and detachability of
each cell species, respectively, and the
aggregability and detachability are
significantly different depending on the cell
species.
(2) Using the conventional methods, it is almost
impossible to regulate the number of cells
constituting the cell clusters or sheets which
controls the size of the cell clusters or
sheets. Since spontaneous aggregation or
detachment of the cells which form the cell
clusters or sheets occurs accidentally and
unexpectedly, the size of the cell clusters or
- 6 - 2~
sheets cannot be regulated and accordingly show
a very broad size distribution.
(3) When the cell clusters or sheets are composed
of a plurality of cell species by the
conventional methods it is difficult to
regulate the population number of each cell
species constituting the clusters or sheets.
The above described problems are crucial if the obtained
cell clusters or sheets are used as an alternative to
tissues or organs in order to repair damaged or diseased
focuses or to evaluate the biological activity of test
substances.
The present invention provides a cell cluster or
sheet comprising a plurality of cell species having a
regulated size and a regulated composition of the cell
species, and a method for preparing a cell cluster or
sheet comprising a plurality of cell species without the
above described problems.
Defi~ition
The term "LCST" is used herein to mean a lower
critical solution temperature which is a transition
temperature of a temperature-responsive polymeric
compound between hydration and dehydration.
~ummar~ of the Inv_ntion
A cell cluster or sheet in accordance with the
present invention comprises a plurality of cell species
and has a regulated size and a regulated composition of
the cell species.
Another aspect of the present invention is a method
for preparing cell clusters or sheets comprising a
plurality of cell species comprises the steps of:
2~37~
7 --
(i) seeding and culturing a plurality of cell
species on a cell culture substrate comprising
(a) a temperature-responslve polymeric
compound having an LCST lower than the
culture temperature and
(b) a cell adhesive substance at a temperature
higher than said LCST,
~ii) detaching an assembly of said cell species
proliferated on said cell culture substrate
from said cell culture substrate by lowering
said temperature to a temperature below said
LCST, and
(iii) culturing said detached cell assembly in
suspension on a non-adhesive substrate to form
a cell cluster or sheet.
Detailed Description of the Invention
According to the present invention, a cell cluster
or sheet comprising a plurality of cell species can be
obtained. The total cell number of the cluster or sheet
and also the population number of each cell species of
the cluster or sheet can be precisely controlled. The
cell cluster or sheet can be obtained by using a cell
culture substrate comprising a temperature-
responsive polymeric compound having an LCST lower than
the cell culture temperature and a cell adhesive
substance capable of effectively allowing the cells to
attach and proliferate.
The temperature-responsive polymeric compound
solidifies above the LCST and instantly dissolves in an
aqueous solution below the LCST. According to the above
described change of the temperature-responsive polymeric
compound it becomes possible to detach the cells cultured
- 8 -
on the substrate the present invention by lowering the
temperature to below the LCST. However, most of the
cells cannot attach and proliferate on the substrate made
from the temperature-responsive polymeric compound alone.
Therefore, the cell adhesive substance of this invention
is essential. The important feature of this invention is
that if the cells form an assembly on the substrate, the
asseffibly does not disintegrate into individual cells and
does not lose the cell number by the above described
detaching process.
Thus, the method fcr preparing the cell cluster or
sheet of the present invention comprises the steps of:
(i) seeding and culturing a plurality of cell
species on the above described cell culture substrate at
a temperature higher than the LCST,
(ii) detaching an assembly of a plurality of cell
species proliferated from the substrate by lowering the
; temperature to a temperature lower than the LCST, and
(iii) culturing the detached cell assembly on a
non-adhesive substrate to form a cell cluster or sheet.
In step (i), it is possible to seed and culture a
plurality of cell species simultaneously or sequentially.
The separate or sequential seeding and culturing of a
plurality of cell species is preferred. Sequential
seeding and culturing is preferred because the
attachability and proliferation ability on the substrate
are so strongly dependent upon the cell species and the
total cell number and the number of each cell species in
the cultured cell assembly are not as easy to control by
the initial feeding concentration of a plurality of cell
species particularly in the simultaneous seeding and
culturing. The most preferable method is to seed and
culture the second cell species after the first cell
2 ~ ~ ~ rj ~ ~;
g
species has been seeded and cultured to a confluent
stage. Numerous layers of cell species can be applied
using the sequential seeding ancl culturing method. Any
practical number of layers is contemplated by this
invention, but preferably about ten layers, and most
preferably about 2-5 layers. The total cell number and
also the number of each cell species of the finally
obtained cell cluster or sheet can be easily and
precisely controlled by the sequential application
method. The number of the confluent first cells on the
substrate can be easily and precisely counted before
seeding the second cell species using a phase-contrast
microscope or other known means. The number of the
second cell species seeded and cultured subsequently can
be calculated by various methods. The calculation
methods include directly counting the number of second
cells attached on the confluent first cells under a
phase-contrast microscope or measuring the number of
unattached second cell species floating in the culture
medium and subtracting this number from the initial
feeding number of the second cell species.
In addition, in the simultaneous seeding and
culturing all of the cell species directly attach to the
substrate, while in the separate and sequential seeding
and culturing only one species directly attaches to the
substrate. Since detachability from the substrate can
also be strongly dependent upon the cell species, the
latter method is preferred -to the former method.
Furthermore, the confluent first cells fully wrap the
second cells up in a kerchief-like structure during the
detaching step, which effectively prevents the loss of
the cells from the cell assembly.
~4~
-- 10 --
In step (iii), the cell assembly detached in step
(ii) is transformed into a cell cluster or sheet by
suspension culture on a cell non-adhesive substrate. One
of the preferred non-adhesive substrates is a
temperature-responsive polymer compound coating.
The cell species which can he employed in the
present invention include fibroblasts~ hepatocytes,
endothelial cells, islet cells, keratinocytes, bone
cells, nerve cells, muscle cells, kidney cells, brain
cells, etc. but in the present invention the cell species
are not limited to these species.
Examples of suitable temperature-responsive
polymeric compounds having an LCST lower than the culture
temperature which can be used in the present invention
are poly-N-substituted (meth)acrylamide derivatives and
their copolymers, polymethylvinylether, polyethylene
oxide, etherized methylcellulose, and partially
acetylated polyvinyl alcohol. Of these preferred
compounds, more preferred are poly-N-substituted
acrylamide derivatives, poly-N-substituted methacrylamide
derivatives and their copolymers.
Preferred examples of such temperature-responsive
polymeric compounds in the present invention are listed
below, but this invention is not limited to these
examples. The LCSTs of these polymers rise with the
sequence of polymers listed below.
Poly-N-acryloyl piperidine, poly-N-n-propyl
methacrylamide, poly-N-isopropyl acrylamide, poly-N,
N-diethyl acrylamide, poly-N-
isopropylmethacrylamide, poly-N-cyclopropyl
acrylamide, poly-N-ethyl acrylamide.
-- 1 1 --
The above described polymers may be homopolymers or
copolymers with other monomers. Any hydrophilic monomers
or hydrophobic monomers can be used as the monomers for
copolymerization. Generally speaking, copolymerization
with a hydrophilic monomer will raise the LCST, and
copolymerization with a hydrophobic monomer will lower
the LCST. With a proper selection of monomers, a
copolymer with a desired LCST can be achieved.
Examples of suitable hydrophilic monomers are
N-vinylpyrrolidone, vinylpyridine, acrylamide,
methacrylamide, N-methyl acrylamide, hydroxyethyl
methacrylate, hydroxyethyl acrylate, hydroxymethyl
methacrylate, hydroxymethyl acrylate, acrylic acid,
methacrylic acid, vinyl sulfonic acid, styrylsulfonic
acid their salts and N,N-dimethylaminoethyl methacrylate,
N,N-diethylaminoethyl methacrylate, N,N-dimethylamino-
propyl acrylamide and their salts, but the present
invention is not limited to these compounds.
Examples of suitable hydrophobic monomers are
acrylate derivatives and methacrylate derivatives such as
ethyl acrylate, methyl methacrylate and glycidyl
methacrylate; N-substituted alkyl (meth)acrylamide
derivatives such as N-n-butyl (meth)acrylamide;vinyl
chloride, acrylonitrile, styrene and vinyl acetate,
but the present invention is not limited to these
compounds.
The molecular weight of the temperature-responsive
polymeric compound which can be employed in the present
invention is preferably at least about 1.0 x 105 and more
preferably higher than about 1.0 x 105. The molecular
weight herein means a number average molecular weight
obtained form the viscosity. For example, the
relationship between the number average molecular weight
~4~6
- 12 -
(Mn) of poly-N-isopropyl acrylamide and its intrinsic
viscosity [~] can be represented by the following
equation [Ito, S. and Geromino, R. T., Sen'i Kobushi
Zairyo Kenkyusho Mokoku (1988) 159:23];
[~] = 9.59 x 10 5 Mn~~
(in tetrahydrofuran solution at 27C).
Examples of the cell adhesive substances which can
be employed in the present invention are extracellular
matrix components, gelatin, lectins, anchorage
oligopeptides which are the binding sites of anchorage
proteins such as fibronectin, adhesive proteins isolated
from shellfish and positively charged polymers. The
extracellular matrix components are the substances
existing among cells within a living body and include
collagen, fibronectin, laminin, vitronectin,
proteoglycan, glycosaminoglycan and thrombospondin.
Examples of such positively charged polymers include
polylysin, polyhistidine, protamine sulfate,
polydimethylaminoethyl acrylate or methacrylate,
polydiethylaminoethyl acrylate or methacrylate,
polydimethylaminoprophyl acrylate or methacrylate,
polyethyleneimine and po~yvinylpyridine.
The cell culture substrate which can be used in the
method of the present invention typically comprises a
coating of a temperature-responsive polymeric compound
having an LCST lower than the culture temperature and a
cell adhesive substance formed on a supporting material.
Such a substrate is prepared by coating an aqueous
solution of a mixture of the temperature- responsive
polymeric compound and the cell adhesive substance on all
or part of the surface of the supporting material at a
temperature below the LCST and drying the coating thus
obtained. The substrate can also be prepared by dipping
2 ~
- 13 -
the supporting material into an aqueous mixture solution
of the temperature-responsive polymeric compound and the
cell adhesive substance at a temperature below the LCST
and drying the coating thus obtained.
Further, the substrate used in the present invention
can be prepared by forming a layer of the temperature-
responsive polymeric compound on a supporting material
and then forming a layer of the cell adhesive substance
on the layer of the temperature-responsive polymeric
compound. Also, the substrate used in the present
invention can be prepared by forming a layer of the cell
adhesive substance on a supporting material and then
forming a layer of the temperature-responsive polymeric
compound on the layer of the cell adhesive substance.
The mixing weight ratio of the temperature-
responsive polymeric compound to the cell adhesive
substance which can be employed in the present invention
is typically from about 1:0.01 to about 1:3. This ratio
varies depending on the type of the cell adhesive
substance used and the cell species used.
The thickness of the coating after it is dried is at
least about 0.2 ~m, preferably at least 0.5 ~m and more
preferably at least about 1.0 ~m. When the thickness is
below about 0.2 ~m, the cell detachability remarkably
worsens and it takes a very long time for the detachment
of the cells and as a result, the cell functions are
rendered unstable.
The supporting material which can be employed in the
present invention is preferably transparent or
translucent, and is preferably of glass or of plastic.
Exemplary plastics include polystyrene, polycarbonate,
polymethyl methacrylate, polypropylene, polyethylene,
polyester, polyamide, polyvinylidene fluoride,
7 ~ ~
- 14 -
polyoxymethylene, polyvinylchloride, polyacrylonitrile,
polytetrafluoro-ethylene and polydimethylsiloxane.
There is no particular limitation on the shape of
the supporting material, and it can take various shapes
such as a dish, plate, film or sheet.
The examples which follow are given for illustrative
purposes and are not meant to limit the invention
described herein. The following abbreviations have been
used throughout in describing the invention.
LCST - lower critical solution temperature
NIPAAm - N-isopropyl acrylamide
PNIPAAm - poly-N-isopropyl acrylamide
PBS - phosphate buffered saline
DMEM - Dulbecco's modified Eagle's medium
FBS fetal bovine serum
HEPES - N-2-hydroxyethylpiperadine-N'-2-
ethanesulfonic acid
HE - hematoxylin-eosin
ABC - avidin-biotin-peroxidase complex
DAB - 3,3'-diaminobenzidine tetrahydro
chloride
Example 1
N-isopropyl acrylamide monomer (herein "NIPAAm", a
product of Eastman Kodak Co.) 50 g was dissolved in
benzene 500 ml, and 2,2'-azobisisobutyronitrile 0.2 g was
used as the polymerization initiator to conduct
polymerization at 60C for 12 hours in a stream of
nitrogen gas with constant agitation. The polymer
precipitated in benzene, was decanted, and the
precipitate was dissolved in tetrahydrofuran and then
purified by precipitation with ethyl ether to obtain
poly-N-isopropyl acrylamide (herein "PNIPAAm"). The
PNIPAAm thus prepared had a number average molecular
~ ~ L~ 3 7 ll 6
-- 15 --
weight of 2.0 x 106, and the LCST of the 0.5% (w/v)
aqueous PNIPAAm solution and the LCST of the 1.0% (w/v)
~NIPAAm phosphate buffer solution (herein "PBS") were
determined by turbidimetry, and they were about 32C and
about 29C, respectively. Using the 0.5% (w/v) aqueous
type I collagen solution derived from pepsinized calf
skin (sterilized, a product of Kohken K.K.) a mixed
solution containing 0.25% (w/v) PNIPAAm and 0.25% (w/v)
collagen as the final concentration was prepared. Using
two types of templates having holes of about 9 mm and
15 mm in diameter, the above described mixed solution was
aseptically coated onto a commercial hydrophobic dish
having a diameter of 60 mm (Falcon) and the coated
culture substrates (Type I substrate) having a surface
area of about 0.6 and 1.8 cm2, respectively were prepared.
The weight mixing ratio of collagen to PNIPAAm in the
coating was 1:1 and the coating thickness was about 2 ~m.
In addition, Type II substrate was prepared by merely
coating the solution containing 0.5% (w/v) PNIPAAm on a
commercial tissue culture dish having a diameter of 35 mm
(Falcon) at the thickness of about 2 ~m.
Human dermal fibroblasts were obtained and
maintained according to the reported method
(Yoshizato, K., et al., Biochem. Biophys. Acta (1980)
627:23). Primary parenchymal hepatocytes were isolated
from 5 week-old male Sprague-Dawley rats weighing about
150 g (Charles River Japan, Inc.) by the perfusion method
of the liver in situ with 0.05% (w/v) collagenase.
Pre-warmed (37C) human dermal fibroblast suspension in
the culture medium [Dulbecco's modified Eagle's medium
(herein "DMEM") containing 10% (w/v) fetal bovine serum
(herein "FBS"), 20mM N-2-hydroxyethylpiperadine-
N'-2-ethanesulfonic acid (herein "HEPES"), 100 units/ml
7 l~ ~
- 16 -
penicillin and 100 mg/ml streptomycin] was seeded on the
pre-warmed (37C) above described Type I substrates of
0.6 and 1.8 cm2 in surface area at the initial cell
density of about 4.G x 104/cm2, respectively. After
3 days of culturing at 37C ln a humidified atmosphere of
5% by volume of C02 and 95~ by volume of air, the
fibroblasts proliferated to a confluent stage. Here, the
confirmation of the confluent state and the measurement
of the confluent fibroblast density were carried out by
0 an inverted phase-contrast microscopic study. Then the
pre-warmed (37C) above described rat primary parenchymal
hepatocyte suspension in the culture medium was seeded on
each of the above-described confluent fibroblast
monolayers at cell densities of about 1.0 x 104 and 5.0 x
104/cm2, respectively. After 60 minutes of co-culturing,
it was confirmed by an inverted phase-contrast microscope
that more than 90% of the seeded hepatocytes attached to
the fibroblast monolayer. The culture dishes were
brought out from the 37C-incubator to an ambient
temperature (about 25C~ and left to stand for about
5 minutes. By this procedure, the hepatocyte-attached
fibroblast monolayer was completely detached from the
Type I substrate as a cell assembly. The detached cell
assembly was rinsed twice with chilled PBS and once with
chilled culture medium in order to avoid contamination of
PINPAAm and collagen dissolved in the culture medium, and
then transferred into pre-warmed (37) culture medium on
the above described Type II substrate. The cell cluster
formation and culture were carried out at 37C in a
humidified atmosphere of 5% by volume of CO2 and 95% by
volume of air on the Type II substrate and the culture
medium was changed every three days.
2 ~ l`? ~
- 17 -
The number of fibroblasts and hepatocytes in the
cell clusters was measured by the following methods. The
4-day cultured cell clusters were fixed in 10% formalin
neutral buffer solution for 60 minutes at 4C. They were
dehydrated and embedded in paraffin wax. Sections were
cut in the vicinity of its center at a thickness of 2 ~m.
After removal of the wax, hematoxylin-eosin (herein "HE")
staining by the standard procedure was carried out. For
the immunoperoxidase staining, dewaxed sections were
immersed in methanol containing 0.3% by weight of H202 for
30 minutes to remove endogenous peroxidase activity.
Then the sections were incubated with goat serum for
60 minutes, with rabbit IgG fraction against rat albumin
(Cappel, Organon Teknika Corporation) (1:500 dilution in
PBS) for 30 minutes, with biotinylated goat antibody
against rabbit IgG for 30 minutes, and with
avidin-biotin-peroxidase complex (ABC) (Vector
: Laboratories, Inc.) for 30 minutes in a moist chamber at
37C. Bound peroxidase was detected by incubation with
0.5 mg/ml DAB (3,3'-diaminobenzidine tetrahydrochloride)
(Dojindo Laboratories) containing 0.01% by weight of H2O2
for 2 minutes. The sections were counterstained in
hematoxylin and dehydrated before mounting.
The number of fibroblasts and hepatocytes was
calculated from the micrographs of the sections stained
with HE and indirect immunoperoxidase as described above,
respectively. In addition, the diameter of all the
clusters was measured from the phase-contrast
micrographs.
The number of hepatocytes and fibroblasts in all the
clusters and the size of the cell clusters are shown in
in Table I.
-- 18 --
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~ ~ _ a~ o ~ o
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-3
~ o~
u~ a) ~ ~ r~ ~
U~ O I`OU i`
C ,~ ~ ,~
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C E ~
I J Q ul u~
H C I Q r-l +l +l +l +I X
~ ~_ ~ ~ ~D O
~ I~ t) ~
, ,-
,~ '~ ~ O O O O
~ ~ t) ~1 ~ ~ In ~
3~
U: ~ qo
~ h
~ 0~
Z ~ - .
a~ ~ a
t_ t) H -- O O H r~ ~
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o æ
Z ,~
I
~Q~7l~
-- 19 --
Table I shows that the number of fibroblasts and
hepatocytes in the cell clusters can easily and precisely be
controlled by the surface area of Type I substrate and the
seeding density of hepatocytes, respectively and also that
the diameter of the cell clusters is precisely regulated by
the total cell numb~rs in the method of this invention.
Example 2
Pancreatic islets were isolated from WKA adult rats
weighing about 300 g by the method of Lacy (Lacy, P. E.~ et
al., DI~3ETES (1967) 16:35). The diameter of the obtained
islets ranged from 200 to 500 ~m.
Type I substrate was prepared by coating the same mixed
solution containing 0.25% (w/v) PNIPAAm and 0.25% (w/v)
collagen as used in Example 1 onto the commercial tissue
culture dish having a diameter of 35 mm (Falcon). The
weight mixing ratio of collagen to PNIPAAm in the coating
was 1:1 and the coating thickness was about 2 ~m. Also
Type II substrate was prepared by the same method as in
Example 1.
The pre-warmed (37C) human dermal fibroblast
suspension in the same culture medium as in Example 1 was
seeded on the pre-warmed (37C) above described Type I
substrate at an initial cell density of about 4.0 x 104/cm2.
After 4 days of culturing at 37C in a humidified atmosphere
of 5% by volume of CO2 and 95% by volume of air, the
fibroblasts proliferated to a confluent stage. Here, the
confirmation of the confluent state and the measurement of
the confluent fibroblast density were carried out by an
inverted phase-contrast microscopic study. Then, a few
pieces of the above described islets suspended in the
3 r~ ~ ~
- 20 -
pre-warmed (~7OC) DMEM were seeded on the above described
fibroblast monolayer. After 10 hours, it was confirmed by
an inverted phase-contrast microscope that the seeded islets
fully attached onto the fibroblast monolayer. Then, Type I
substrate was brought out from the 37C-incubator to an
ambient temperature (about 25C). It was confirmed by an
inverted phase-contrast microscope that by this procedure
the islet-attached fibroblast monolayer gradually detached
from the Type I substrate and the detached monolayer
aggregated wrapping up the islets on it. In this detaching
process, no dissociation of the islets from the fibroblast
monolayer was recogni~ed. The cell assembly made from
fibroblasts and islets thus obtained was washed 2 to 3 times
with PBS and then transferred into the pre-warmed culture
medium on the above described Type II substrate and cultured
in suspension changing the culture medium every 4 days.
This culture process enabled the cell assembly to transfer
to a complete cell cluster. After 14 days of culturing on
the Type II substrate, the cell cluster was fixed in 10%
formalin neutral buffer solution, dehydrated and embedded in
paraffin wax. Sections were cut in the vicinity of its
center at a thickness of about 2 ~m. After removal of the
wax, the HE staining by the standard procedure was carried
out. The staining clearly showed that the seeded islets
~5 were completely encapsulated with the fibroblasts and the
cell cluster obtained was composed of the islets and the
fibroblasts.
Example 3
Using the same method as in Example 2, the cell cluster
composed of one islet having a diameter of about 250 ~m and
~0~8~
- 21 -
human dermal fibroblasts was prepared. The cell cluster was
cultured i~ DMEM containing 10% (w/v) FBS at 37~C in a
humidified atmosphere of 5% by volume of CO2 and 95% by
volume of air on the Type II substrate by changing the
culture medium every 4 days. The amount of insulin secreted
from the cell cluster into the culture medium was measured
by the standard immunoassay. Even after 40 days of
culturing, the amount of insulin secretion, about 33
~U/islet/day was observed. In addition, after 2 months of
culturing, the cell cluster was observed by the above
described HE staining and as a result, no necrosis was seen
in the islet encapsulated with the fibroblasts. As a
control, only an islet of about 350 ~m in diameter was
cultured on the Type II substrate under the same conditions
as described above without the fibroblasts. After 10 days
of culturing, the cecretion rate of insulin reduced to about
4~U/islet/day and the HE staining demonstrated the
necrotized inside of the islet after 2 wee~s of culturing.
These findings suggest that the formation of the cell
cluster with the fibroblasts effectively maintains the islet
viability and function for a long period of time.
Example 4
Islet cells were prepared from the same islets as in
Example 2 by the reported method (Ono, J., et al.,
Endocrinol. ~apan. (1977) 24:265). Instead of the islets,
the cell cluster of the islet cells and the fibroblasts was
prepared by the same method as in Example 2. As the result
of the HE staining, it was found that the islet cells were
fully encapsulated with the fibroblasts.
2 ~ L~
- 22 -
Example 5
The pre-warmed (37C) human dermal fibroblast
suspension in the same culture medium as used in Example 1
was seeded on the pre-warmed (37C) Type I substrate as used
in Example 1 at an initial cell density of about 4.0 x
104/cm2. After 3 days of culturing at 37C in a humidified
atmosphere of 5% by volume of C02 and 95% by volume of air,
the fibroblasts proliferated up to a confluent stage in a
monolayer. Then a pre-warmed (37C) human umbilical vein
endothelial cells (Sanko Junyaku Co., Ltd.) suspension in
the above described culture medium was seeded on the above
described fibroblast monolayer at a cell density of about
5.0 x 10~/cm2. After 60 minutes, most of the seeded
endothelial cells attached to the fibroblast monolayer.
Then, the Type I substrate was brought out from the 37C
incubator to an ambient temperature (about 25C). It was
confirmed by an inverted phase-contrast microscope that by
this procedure the endothelial cells-attached fibroblast
monolayer was gradually detached from the Type I substrate
and the detached monolayer aggregated wrapping up the
endothelial cells on it. In this detaching process,
significant dissociation of the endothelial cells and
fibroblasts was not observed.
The cell assembly made from fibroblasts and endothelial
cells thus obtained was washed 2 to 3 times with PBS and
then transferred into the pre-warmed culture medium on the
above described Type II substrate and cultured in suspension
changing the culture medium every 4 days. This culture
process enabled the cell assembly to transfer to a complete
cell cluster. After 14 days of culturing on the Type II
substrate, the cell cluster was fixed in 10% formalin
- 23 -
neutral buffer solution, dehydrated and embedded in paraffin
wax. Sections were cut in the vicinity of its center at a
thickness of about 2 ~m. After the wax was removed, the HE
staining by the standard procedure was carried out. The
staining clearly showed that the seeded endothelial cells
were completely encapsulated with the fibroblasts and the
obtained cell cluster was composed of endothelial cells and
fibroblasts.
