U.S. patent application number 10/108471 was filed with the patent office on 2002-11-28 for cellular screening substratum and manufacturing process for it, and method and apparatus for cellular screening with it.
Invention is credited to Matsuda, Ryoichi, Miyazaki, Takeshi, Nishiguchi, Kenji, Watanabe, Kohei.
Application Number | 20020177221 10/108471 |
Document ID | / |
Family ID | 18951027 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177221 |
Kind Code |
A1 |
Nishiguchi, Kenji ; et
al. |
November 28, 2002 |
Cellular screening substratum and manufacturing process for it, and
method and apparatus for cellular screening with it
Abstract
The present invention provides cellular screening substrata
which can be formed in simple processing steps. The cellular
screening substrata can be formed which are characterized in that
plural cellular screening substances are positioned and immobilized
at predetermined positions on a base by micro-droplet discharging
means, and plural areas having different cellular screening
functions are formed thereon.
Inventors: |
Nishiguchi, Kenji;
(Kanagawa, JP) ; Miyazaki, Takeshi; (Kanagawa,
JP) ; Matsuda, Ryoichi; (Tokyo, JP) ;
Watanabe, Kohei; (Tokyo, JP) |
Correspondence
Address: |
FITZPATRICK CELLA HARPER & SCINTO
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
US
|
Family ID: |
18951027 |
Appl. No.: |
10/108471 |
Filed: |
March 29, 2002 |
Current U.S.
Class: |
435/287.2 ;
427/2.11; 435/6.11 |
Current CPC
Class: |
B01J 2219/00585
20130101; G01N 2510/00 20130101; B01J 2219/00612 20130101; G01N
33/5008 20130101; B01J 19/0046 20130101; B01J 2219/00378 20130101;
B01J 2219/00596 20130101; B01J 2219/00659 20130101; B01J 2219/00605
20130101; C12M 41/38 20130101; C40B 60/14 20130101; B01J 2219/00743
20130101; C12M 25/06 20130101; B01J 2219/00497 20130101; B01J
2219/00527 20130101; B01J 2219/00707 20130101; G01N 33/5029
20130101 |
Class at
Publication: |
435/287.2 ;
427/2.11; 435/6 |
International
Class: |
C12Q 001/68; C12M
001/34; B05D 003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 2001 |
JP |
097218/2001 (PAT. |
Claims
What is claimed is:
1. A cellular screening substratum, wherein plural cellular
screening substances are positioned and immobilized at
predetermined areas on a base by micro-droplet discharging means to
form plural areas having different functions for cellular
screening.
2. The cellular screening substratum according to claim 1, wherein
said plural areas comprise plural areas having a different
combination of the immobilized cellular screening substances each
other.
3. The cellular screening substratum according to claim 1, wherein
said plural areas comprise plural areas having a different density
of the immobilized cellular screening substances each other.
4. The cellular screening substratum according to claim 1, wherein
each area or each area group of two or more areas is formed within
a sunken part.
5. The cellular screening substratum according to claim 1, wherein
each area or each area group of two or more areas is surrounded by
a rising wall-shaped structure.
6. A process for manufacturing a cellular screening substratum,
comprising the steps of positioning each of cellular screening
substances at predetermined areas on a base by micro-droplet
discharging means; and immobilizing the cellular screening
substances at each area on the substratum.
7. The process for manufacturing a cellular screening substratum
according to claim 6, wherein the micro-droplet discharging means
is adopting thermal ink-jet method.
8. The process for manufacturing a cellular screening substratum
according to claim 6, wherein the micro-droplet discharging means
is adopting piezoelectric ink jet method.
9. The process for manufacturing a cellular screening substratum
according to claim 6, wherein said immobilization step further
comprises the step of applying external immobilization energy.
10. A method for cellular screening using a cellular screening
substratum according to claim 1, comprising the step of culturing
cells in a culture medium in con tact with the areas of the
immobilized screening substances on the cellular screening
substratum.
11. The method for cellular screening according to claim 10,
comprising the step of adding a substance required for screening
said cells to the culture medium in contact with the areas of the
immobilized screening substances.
12. The method for cellular screening according to claim 10,
wherein the areas of the immobilized screening substances are in
contact with the flow of the culture medium.
13. The method for cellular screening according to claim 10 further
comprising the step of observing morphological change of the
cells.
14. The method for cellular screening according to claim 13,
wherein the cells are stained upon evaluation.
15. The method for cellular screening according to claim 10,
comprising the step of measuring quantitatively a substance which
has been synthesized within the cells.
16. The method for cellular screening according to claim 10,
comprising the step of measuring quantitatively a substance which
has been incorporated into the cells.
17. The method for cellular screening according to claim 15,
comprising the step of measuring quantitatively the substance by
determining the amount of radiation, fluorescence, light emission
or absorbance.
18. The method for cellular screening according to claim 16,
comprising the step of measuring quantitatively the substance by
determining the amount of radiation, fluorescence, light emission
or absorbance.
19. An apparatus for cellular screening using a cellular screening
substratum according to claim 1, wherein said apparatus comprises
means for culturing the cells in a culture medium in contact with
the areas of the immobilized screening substances on the cellular
screening substratum.
20. The apparatus for cellular screening according to claim 19,
further comprising at least one of means for observing
morphological changes of cells, means for measuring quantitatively
a substance which has been synthesized within the cells, and means
for measuring quantitatively a substance which has been
incorporated into cells by said culturing means.
21. The apparatus for cellular screening according to claim 19,
further comprising means for manufacturing the cellular screening
substratum.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to cellular screening
substrata capable of being used for identifying substances
contributing to at least one of cell adhesion, proliferation,
differentiation, survival, maintenance of undifferentiated state,
and apoptosis, to their manufacturing processes, and to methods and
apparatus for cellular screening using the cellular screening
substrata.
[0003] 2. Related Background Art
[0004] Recently, there have been actively carried out studies on
culture of animal and plant cells under different conditions, or
studies on products of particular cultured cells. Specifically,
investigations have been made in various fields to produce,
utilizing specific cellular activities, substances which are
impossible to synthesize artificially or otherwise quite difficult
to synthesize.,
[0005] Also, there have been carried out studies to identify
substances having an effect on cell proliferation and
differentiation to proliferate or differentiate desired cells
according to intended applications. With rapid progress in cell
technology and medical engineering, attention has focused on
microbiosensors or artificial organs using cells, and furthermore
on neurocomputers and the like, and active research efforts have
been made for these applications.
[0006] In order to utilize cells in vitro as stated above, however,
it is indispensable to position cells as desired and to control
their proliferation, differentiation, and production of substances.
Mechanisms that control cell positioning, proliferation,
differentiation, and production of substances, however, have not
been sufficiently elucidated yet. Thus, it is quite difficult to
culture cells controlling these cellular functions, which is one of
the main obstacles to researches and developments utilizing cells
as described above.
[0007] As an attempt to regulate the cell positioning, U.S. Pat.
No. 5,108,926 discloses a method employing an ink jet printer to
form a pattern of a cell adhesive protein on which cells are grown.
By this method, cells can be cultured on the surface of the pattern
where a cell adhesive protein was applied, but it is impossible to
control their proliferation/differentiation and production of
substances to screen the cells.
[0008] In an article (Proteins, Nucleic Acids and Enzymes, 45-5,
727-734 (2000), cell growth factors that effect proliferation and
differentiation of cells were immobilized onto a support using a
photolithography technique, and their effects on proliferation and
differentiation of cells were studied. The substrate on which the
cell growth factors had been immobilized, however, was not used as
means for cellular screening. In addition, photolithographic
procedures waste biological substances that exist only in small
amounts in the body, and necessitate repeating the processes of
exposure and development, complicating the production steps.
[0009] WO97/45730 proposes a method of screening cells by
immobilizing a substance that influences cell adhesiveness onto a
substrate. In this method, a reactive functional group provided on
the substratum and a cell adhesive material is immobilized through
a divalent crosslinking reagent. This method employs
photolithography to bind the reactive functional group to the cell
adhesive material. Thus, this method has problems as described
above. In addition, when two or more cell adhesive materials are
immobilized, it is almost impossible to avoid the binding of the
material which has been already immobilized and a material to be
newly immobilized at undesired locations through a divalent
crosslinking reagent, and therefore it is very difficult to
position cell adhesive materials on desired locations. Further,
this method does not include immobilization of substances that
affect cell proliferation, differentiation or production. In
addition, according to this method, cells are fixed in wells
through the cell adhesive materials, and screening is carried out
by detecting substances produced by the cells during culture of the
cells. Thus, this method is not a method for screening substances
which have an effect on proliferation and differentiation, and
furthermore production of substances, as in the present
invention.
SUMMARY OF THE INVENTION
[0010] The present invention aims to provide a substratum for
cellular screening that can solve the problems in the
above-described prior arts, and can be prepared by simple
procedures, and to provide a manufacturing process thereof, whereby
providing basic techniques for further progress of researches in
cell technology etc., and for manufacturing various devices
utilizing cells.
[0011] Furthermore, the present invention aims to provide a method
for screening substances which have an effect on at least one of
cell adhesiveness, proliferation, differentiation, survival,
maintenance of undifferentiated state, apoptosis, and production of
substances, using cells cultured on such a substratum.
[0012] Results of screening allow identifying factor(s) necessary
for cell proliferation or differentiation, survival, maintenance of
undifferentiated state, apoptosis, or production of substances, and
determining methods for efficient cell culture. In addition, if the
substances to be immobilized are drugs, then it is possible to
evaluate which combination of drugs and what amount exert the most
effective effort on cultured cells. Alternatively, for example, by
using a substratum on which sustained-release capsules of
polyacrylamide gel etc. containing chemicals such as so-called
endocrine disrupting chemicals are immobilized, and allowing
gradual release of the chemicals from the capsules into the culture
medium, it is possible to assess the sensitivity of human to such
chemicals. Furthermore, based on the results of these assessments,
it will be possible to determine a method of diagnosing individuals
for various diseases.
[0013] According to one aspect, the present invention provides a
cellular screening substratum, wherein plural cellular screening
substances are positioned and immobilized at predetermined areas on
a base by micro-droplet discharging means to form plural areas
having different functions for cellular screening.
[0014] In an embodiment, the above-described areas at which
screening substances have been immobilized may contain more than
one area having a different combination of the immobilized cellular
screening substances. In another embodiment, the plural areas at
which screening substances have been immobilized may also contain
more than one area having a different density of the immobilized
cellular screening substances. In still another embodiment, each
area or each area group of two or more areas may be formed within a
sunken portion. Each area or each area group of two or more areas
may be surrounded by a rising wall-shaped structure.
[0015] In another aspect, the present invention includes A process
for manufacturing a cellular screening substratum, comprising the
steps of positioning each of cellular screening substances at
predetermined areas on a base by micro-droplet discharging means;
and immobilizing the cellular screening substances at each area on
the substratum.
[0016] In one embodiment, droplet-discharging means by a thermal
ink jet method, or a piezoelectric ink jet method is used.
[0017] In one embodiment, external immobilizing energy is applied
to immobilize a culture controlling substance on the base.
[0018] In still another aspect, the present invention provides a
method for cellular screening using a cellular screening substratum
according to claim 1, comprising the step of culturing cells in a
culture medium in contact with the areas of the immobilized
screening substances on the cellular screening substratum.
[0019] In one embodiment, t is possible to add one or more
substances required for cellular screening to the culture medium in
contact with the areas of the immobilized screening substances. It
is also possible to carry out cell culture under conditions in
which the areas of the immobilized screening substances are in
contact with the flow of the culture medium, for example, by
perfusing the culture medium.
[0020] Screening can be performed, based on the following. In this
case, cells may be stained in advance.
[0021] 1) Evaluation of morphological changes of cells in a desired
area.
[0022] 2) Quantitative measurement of a substance synthesized by
the cells in a desired area on the substratum.
[0023] 3) Quantitative measurement of a substance incorporated into
the cells in a desired area on the substratum.
[0024] 4) Quantitative measurement of a substance by determining
the amount of radiation.
[0025] 5) Quantitative measurement of a substance by determining
the amount of fluorescence.
[0026] 6) Quantitative measurement of a substance by determining
the amount of light emission.
[0027] 7) Quantitative measurement of a substance by determining
the absorbance.
[0028] An apparatus for cellular screening using a cellular
screening substratum as described above, wherein said apparatus
comprises means for culturing the cells in a culture medium in
contact with the areas of the immobilized screening substances on
the cellular screening substratum.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a schematic example of the process for
manufacturing the cellular screening substratum of Example 1;
[0030] FIG. 2 is an example of positioning cellular screening
substances on the cellular screening substratum of Example 1;
[0031] FIG. 3 is an example of positioning cellular screening
substances on the cellular screening substratum of Example 2;
[0032] FIG. 4 is an example of positioning allergens on the
allergen screening substratum of Example 3;
[0033] FIG. 5 is an example of positioning cellular screening
substances on the screening substratum of Example 4;
[0034] FIG. 6 shows a block diagram of the apparatus for cellular
screening according to the claimed invention; and
[0035] FIG. 7 shows an illustration of means for manufacturing a
cellular screening substratum of the apparatus for cellular
screening according to the claimed invention.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention will be now described in detail
below.
[0037] There will be explained one embodiment of the cellular
screening substratum of the present invention. As shown in FIG. 2,
the cellular screening substratum 1 has two or more (four in FIG.
2) substances to be screened with cells (hereinafter referred to as
cellular screening substance or screening substance) 12 positioned
on desired locations (121 to 124), each of the cellular screening
substances 12 being immobilized on the base 11. Immobilization of
two or more cellular screening substances 12 will make it possible
to control at least one of adhesion, proliferation, and
differentiation of cells to a high degree.
[0038] In the present invention, the cellular screening substances
12 refer to culture-controlling substances that effect cell
adhesiveness onto a base 11, proliferation, differentiation,
survival, maintenance of undifferentiated state, apoptosis, or
production of substances, including extracellular matrix proteins,
antibodies having an ability of binding specifically to the cell
surface, cytokines, and other chemical substances.
[0039] Extracellular matrix proteins include, for example,
collagen, elastin, fibronectin, laminin, and others. Cytokines
include cell growth factors and hormones. Cell growth factors
include nerve growth factors (NGFs), epidermal growth factors
(EGFs), fibroblast growth factors (FGFs), and others. Hormones
include insulin, adrenaline, and others.
[0040] Other chemical substances include substances such as
allergens that cause allergy and various chemicals called endocrine
disrupting chemicals. Immobilized cellular screening substances 12
form areas according to the difference in chemical or physical
properties such as types of the cellular screening substances 12
and disposed patterns on the base 11.
[0041] The combination of cellular screening substances 12 can be
different between areas or area groups of two or more areas on the
base 11. This will allow observing the different effect of the
combinations of substances 12 on at least one of cell adhesiveness,
proliferation, differentiation, survival, maintenance of
undifferentiated state, apoptosis, and production of
substances.
[0042] The cellular screening substances 12 can be also represented
at.different densities according to the areas or area groups of two
or more areas on the base 11. This will allow observing in more
detail difference in cell adhesiveness, proliferation,
differentiation, survival, maintenance of undifferentiated state,
apoptosis, or production of substances due to differences in the
density of cellular screening substances 12. Thus, one major
advantage of employing droplet discharging means is that it is
possible that cellular screening substances are readily positioned
onto an immobilization area at a given ratio.
[0043] Immobilization of cellular screening substances 12 onto a
base 11 can be made via a covalent bond, electrostatic attraction
force, or biological affinity. When the cellular screening
substances 12 are immobilized onto a base 11 by a covalent bond,
the substances 12 can be immobilized with strong force, and the
binding is hardly influenced depending on cells, culture medium,
and the like, resulting in stable immobilization on the base
11.
[0044] Now, referring to FIG. 1, an example procedure for
immobilizing a substance 12 onto a base 11, in which insulin is
employed as a substance 12 having an effect on at least one of cell
adhesiveness, proliferation, differentiation, survival, maintenance
of undifferentiated state, apoptosis, and production of
substances.
[0045] First, 4-azidobenzoic acid N-hydroxysuccinimide ester is
introduced into insulin as a linker (see the following scheme).
1
[0046] A solution of the linker-attached insulin 12 thus obtained
is discharged, for example, onto a polyethylene terephthalate (PET)
base 11, using liquid droplet discharging means 13 (an ink jet
printer). Next, when this base is irradiated with light, for
example, UV light, from a light source indicated by 15 in FIG. 1,
and the azido group of the linker is cleaved to form an amide bond
with a carbon atom on the PET base that the insulin 12 is
covalently immobilized on the surface as shown in the following
formula. 2
[0047] In the case of immobilization via electrostatic attraction
force, the immobilization onto the base 11 can be achieved without
chemical treatments, avoiding denaturation of cellular screening
substances 12 due to chemical treatment. When biological affinity
is utilized for immobilization onto a base 11, it is relatively
easy to carry out treatments required for immobilizing cellular
screening substances 12, and thus stable immobilization is
achievable.
[0048] Base 11 can be of any material and of any shape, as long as
the stable immobilization of cellular screening substances 12 can
be obtained. Specifically, glass plates, plastic plates, plastic
sheets, polymer films, papers, and the like can be suitably
employed. Base 11 can be transparent, or light-shielding, or even
colored. In order to immobilize cellular screening substances 12
onto a base 11, or in order to enhance the stability of cellular
screening substances 12 on a base 11, portion or the entire of the
surface of a base 11 can be treated with chemical(s), or by
exposing it to radiation.
[0049] On a base 11, individual areas or area groups of two or more
areas in which cellular screening substances 12 are immobilized may
be sunken or depressed. This can facilitate the positioning of
liquid droplets by micro-droplet discharging means, and in
addition, permits cell culture changing the culture medium for
every area or area group in the same sunken region. Bases having
these depressions can made by die molding of resin materials, by
etching procedures using photolithography techniques or by
others.
[0050] On a base 11, individual areas or area groups of two or more
areas in which cellular screening substances 12 are immobilized may
be surrounded by a wall-shaped structure. This can facilitate the
positioning of liquid droplets by micro-droplet discharging means,
and in addition, permits the cell culture changing the culture
medium in every area or every area group in the same depression.
Such bases having wall-shaped microstructures can be made by
employing photolithography methods etc.
[0051] A cellular screening substratum 1 of this type can be
manufactured as follows (see FIG. 1). A base 11 may be optionally
subjected to the above-described pretreatment first. Specifically,
a base 11 can be subjected to various chemical and physical
treatments such as washing to remove unwanted materials, radiation
including UV light, or corona discharging. In addition, a polymer
material or a silane coupling agent may be applied onto portion or
the entire of the surface of a base 11, if necessary.
[0052] Cellular screening substances 12 are positioned on such a
base 11. For positioning them is employed micro-droplet discharging
mean 13. Here, micro-droplet discharging mean 13 refers to means
capable of discharging liquid droplets having a volume of 100 pl or
less per droplet, including micro-pipettes, micro-dispensers,
discharging devices utilizing an ink jet method. In view of cost of
the discharging device production and micro-droplet discharging
ability, discharging devices utilizing an ink jet method can be
suitably employed. Among ink jet methods, a thermal ink jet method
and a piezoelectric ink jet method can be suitably employed. The
discharging device of thermal ink jet method has advantages that
micro-processing of the discharging port is easy, and it can
dispose cellular screening substances 12 at high density. The
discharging device of piezoelectric ink jet method generates
discharging energy by the displacement of the piezoelectric element
so that thermal stress is not applied to the cellular screening
substances 12 and the substances 12 can be discharged without
impairing their properties.
[0053] Cellular screening substances 12 can be dissolved in an
appropriate solvent. Such a solvent may be any solvent, as long as
it can dissolve cellular screening substances 12 without impairing
their properties. Suitably used is water, preferably ion-exchanged
(deionized) water, or otherwise various buffer solutions is
preferable, because they dissolve cellular screening substances 12
without impairing their properties.
[0054] Aqueous solvents can be also used, if necessary. Aqueous
solvents may be any solvent, as long as it dissolves in water, and
include, for example, alkyl alcohols having up to 14 carbons, such
as methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl
alcohol, n-butyl alcohol, sec-butyl alcohol, and ter-butyl alcohol;
amides such as dimethylformamide and dimethylacetamide; ketones and
keto alcohols such as acetone and diacetone alcohol; ethers such as
tetrahydrofuran and dioxane; polyalkylene glycols such as
polyethylene glycols and polypropylene glycols; alkylene glycols
having an alkylene group containing 2 to 6 carbon atoms, such as
ethylene glycol, propylene glycol, butylene glycol, triethylene
glycol, 1,2,6-hexanetriol, thiodiglycol, hexyleneglycol, and
diethylene glycol; glycerin; lower alkyl ethers of polyhydric
alcohols such as ethylene glycol monomethyl ether, ethylene glycol
monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol
monomethyl ether, diethylene glycol monoethyl ether, diethylene
glycol monobutyl ether, triethylene glycol monomethyl ether,
triethylene glycol monoethyl ether, and triethylene glycol
monobutyl ether; N-methyl-2-pyrrolidone, 2 -pyrrolidone,
1,3-dimethyl-2-imidazoline- , and the like. Among many aqueous
organic solvents as listed above are preferred polyhydric alcohols,
such as diethylene glycol, and lower alkyl ethers, such as
triethylene glycol monomethyl ether.
[0055] Among these solvents, ethanol or isopropyl alcohol, or a
lower alkyl ether of polyhydric alcohols is suitably used because
its addition permits more stable bubble formation in ink on the
thin-film resistor element in the discharging head in the case of a
thermal jet printer.
[0056] In addition to the above-described components, the solution
containing a cellular screening substance 12 can contain
surfactants, antifoaming agents, preservatives, inorganic salts,
organic salts, and others optionally, in order to prepare a
solution with desired physical properties.
[0057] For example, surfactants can be any surfactant, as long as
it dose not exert an adverse effect on cellular screening
substances 12 in storage stability and others, and include, for
example, anionic surfactants, such as fatty acid salts, higher
alcohol sulfuric acid ester salts, liquid fatty oil sulfuric acid
ester salts, and alkylallylsulphonate salts; and nonionic
surfactants, such as polyoxyethylenealkyl ethers,
polyoxyethylenealkyl esters, polyoxyethylenesorbitanalkyl esters,
acetylene alcohol, and acetylene glycol. One or more surfactants of
these can be selected and used as appropriate.
[0058] After cellular screening substances 12 are positioned to
desired positions on a base 11 by micro-droplet discharging means
13, the substances 12 are immobilized on the base 11. In order to
immobilize cellular screening substances 12 on a base 11, the
cellular screening substances 12 or the base 11 may be subjected to
pre-treatment necessary for immobilization. Treatments directed to
the cellular screening substances 12 include: introduction of a
functional group for covalent bonding such as amino, carboxyl,
disulfide, epoxy, carbodiimide, and maleimido groups, or attachment
of electrically chargeable materials for binding via electrostatic
attraction force such as metals and inorganic oxide particulates,
and cationic and anionic macromolecules. In order to achieve
binding through biological affinity, on the other hand, avidin or
biotin molecules, or materials capable of binding through
biological affinity, such as antigen or antibody molecules can be
attached to the substances. Alternatively, the surface of the base
can be coated with a macromolecule or a silane coupling agent to
introduce functional groups, such as amino, carboxyl, disulfide,
epoxy, carbodiimide, and maleimide groups for covalent bonding, or
in order to charge the base surface, a conductive or semiconductive
layer can be formed in advance on the surface, for example, by
using one of metals such as gold, silver, platinum, and iron,
inorganic oxides such as indium tin oxide, titanium oxide, and zinc
oxide, and furthermore conductive macromolecules such as
polyacetylene, polypyrrole, polyaniline, and polythiophene, others.
Otherwise, the surface of a base 11 can be provided with a
substance that can bind to the bioaffinity substance introduced to
the screening substance 12, which includes biotin or avidin,
antibodies or antigens, and protein A having an antibody binding
capability. Introduction of such a substance can strengthen binding
force between the surface of a base 11 and cellular screening
substances 12.
[0059] For immobilization, it is possible to apply energy
externally by exposure to radiation including light, or by heating.
Applying these external energies can promote the binding of the
surface of a base 11 and cellular screening substances 12.
[0060] A cellular screening substratum 1 can be manufactured in the
above-described procedure.
[0061] There will be now described a method of culturing cells on
the cellular screening substratum 1 described above. By culturing
cells on such a substratum, the cells will be cultured under the
influence exerted on their adhesiveness, proliferation and
differentiation, survival, maintenance of undifferentiated state,
apoptosis, or production of substances. Cells are not limited
specifically, and cells of any type can be used. One or more types
of cells can be used for cellular screening. If necessary,
sterilization treatment can be carried out by irradiating a
cellular screening substratum 1 with ultraviolet light or the like,
or by washing it with an alcohol solution before cell culture.
These treatments allow preventing the culture from inhibition due
to undesired microorganisms and the like. Although the culturing of
cells can be performed by immersing the entire cellular screening
substratum 1 into a culture medium, cells can be cultured under the
influence exerted on their adhesiveness, proliferation and
differentiation, survival, maintenance of undifferentiated state,
apoptosis, and furthermore production of substances, so long as the
region having the cellular screening substances immobilized therein
is immersed in the culture medium.
[0062] Further, during culturing cells on the cellular screening
substratum 1, or after culturing cells for a given period of time,
it is possible to add a desired substance or substances to-the
culture medium for a desired region. This may result in alteration
of cell proliferation and differentiation, survival, maintenance of
undifferentiated state, apoptosis, or production of substances, and
the adhesiveness to the substratum. It is also possible to add a
desired substance or substances such as indicators to a desired
region to facilitate screening after culturing cells.
[0063] During culturing cells on the cellular screening substratum
1, or after culturing cells for a given period of time, it is
possible to remove a population of cultured cells from the
substratum. When this is done, the substratum from which the
cultured cells have been removed can be reused, and the removed
population of cultured cells can be also used as artificially made
living tissues or their portions. In specific procedures, a
cellular screening substratum after culture can be trypsinized to
remove a population of cultured cells, thereby reusing the
substratum. This reuse of the substratum is one of the advantages
provided by immobilizing cellular screening substances on the base,
since cells cannot take in such cellular screening substances into
the metabolite system. In addition, if a polymer such as
poly(N-isopropylacrylamide) of which solubility in water varies
with temperature is applied onto the substratum in advance, and
after cell culture the temperature is reduced to about 30.degree.
C. or lower, the change of the hydration state on the polymer
surface permits removal of a population of the cultured cells.
Thus, the cell population can be utilized for living tissues and
the like.
[0064] Next, there will be described a method of culturing cells on
the above-described cellular screening substratum 1, thereby
cellular screening and substances immobilized on the substance. As
screening means can be utilized methods by which morphological
changes in cells cultured on the above-described cellular screening
substratum 1 are observed. One can employ any of microscopes,
including optical microscopes, such as scanning electron
microscopes, transmission electron microscopes, scanning probe
microscopes, and fluorescence microscopes, as long as cell
morphology can be observed. The cellular screening substratum on
which cells have been cultured is placed at the observing position
of the above-mentioned microscope, and cell morphology is observed
with the microscope. Screening can be performed only by observing
cell morphology under a microscope, and thus evaluation can be
conducted by simple methods. Upon evaluation, the cells can be
stained. Staining cells can facilitate evaluation with a microscope
in the case where cells have been grown to high densities or fused
to each other due to differentiation to form polykaryotic
cells.
[0065] Besides morphological observation, one can utilize, as
screening means, quantifying a substance produced by or
incorporated into cells, during the course of or as results of the
adhesion of cells to the substratum or the undergoing of
proliferation and differentiation of cells. If a subject to be
quantified is not measurable directly, an alternative substance can
be quantified. Specifically, genetic engineering can be used to
integrate a gene of a quantitatively measurable protein in the
vicinity of the gene of a desired subject protein to be quantified,
so that the desired protein can be quantitatively determined by
quantifying the quantitatively measurable protein. By determining
these substances, it is possible to investigate in detail what
intracellular changes are caused by the substances immobilized on
the substratum, leading to the elucidation of signal transduction
mechanisms within the cells. In the case where evaluation is
conducted with a substance incorporated into the cells, a
measurable indicator can be provided in advance to a substance
which will be incorporated, allowing quantification with relative
ease.
[0066] Quantifying of these substances involves methods of
measuring the amount of radiation emitted from a radioactive
compound, methods of measuring the amount of fluorescence emitted
from a substance labeled with a fluorescent substance, and
furthermore methods of measuring the amount of light emitted from a
light-emitting substances, and methods of measuring the absorbance
of a dye.
[0067] In methods of measuring the amount of radiation emitted from
a radioactive compound, a method employing a compound substituted
with a radioisotope element abundant in the body, such as hydrogen,
carbon, nitrogen, phosphorus, and sulfur, to measure the radiation
emitted from the compound is highly sensitive. In addition, since
such a compound has the same chemical properties as those of cold
compound, activities of the cellular metabolism is not affected,
thus permitting observation of similar phenomena to those within
the living body.
[0068] Labeling with a fluorescent substance is relatively easy,
and since such fluorescent substances are low molecular weight
compounds, they will exert a slight effect on activities of the
cellular metabolism. In addition, when a substance produced by the
cells is quantified by quantitative methods using an
antigen-antibody reaction, various antibodies labeled with
fluorescent substances are commercially available and have high
sensitivities for measurement. Thus, evaluation with fluorescent:
measurements is effective.
[0069] In methods of measuring the amount of light emitted from a
light-emitting substance, it is possible to measure the emitted
light amount at high sensitivities, so that significantly small
changes can de detected. In the case where a gene has been
identified which is expressed accompanying with adhesion,
proliferation, differentiation, or production of substance caused
by the cellular screening substances, a firefly luciferase gene or
the like can be introduce near that gene, and amounts of the
luciferase produced in conjunction with the gene expression can be
measured by means of the amount of light yielded by addition of ATP
and luciferin. In this way, the effects caused by the screening
substances can be evaluated by virtue of the amount of light
emission.
[0070] In methods of measuring the absorbance of a dye, it is
possible to amplify the absorbance of a dye, in combination with
enzyme reactions and the like, thereby measuring quantitatively a
substance occurring in very small amounts.
[0071] The following describes an apparatus for culturing cells on
the above-described cellular screening substratum and for screening
cells and substances immobilized on the substratum. This aspect of
the present invention is characterized by positioning and
immobilizing two or more cellular screening substances in desired
areas on a base by micro-droplet discharging means, and culturing
cells in a culture medium in contact with the areas of the
immobilized cellular screening substances on the cellular screening
substratum having plural areas of different functions, and
furthermore it can involve means of manufacturing the
above-described cellular screening substratum, or at least one of
means for evaluating changes in cell morphology cultured by the
culturing means, means for measuring quantitatively a substance
synthesized within the cells, and means for measuring
quantitatively a substance incorporated into the cells.
[0072] FIG. 6 shows a block diagram of the apparatus according to
the claimed invention. A base (600) is supplied from the base
supplying chamber (601) of the apparatus. In the
screening-substance applying chamber (602), screening substances
are applied on the base with micro-droplet discharging means, and
then in the immobilizing chamber (603), immobilized by exposing it
to light or heat to manufacture a cellular screening substratum.
Next, the resulting substratum is transferred into the culturing
chamber (604) and cells are cultured on the substratum by the
above-described method, followed by cellular screening in the
detection chamber (605), by observing morphological changes of
cells, cell adhesiveness, proliferation and differentiation,
survival, maintenance of undifferentiated state, apoptosis, or
production of substances, or by the above-described quantifying
means.
[0073] Chambers 601 to 603 can be represented, for example, by
apparatus as shown in FIG. 7, wherein 710 designates a
micro-droplet discharging apparatus. Substrata 600 are set in the
stocker 711, and a substratum is transferred to the belt conveyor
713 through the transferring machine 712 and sent to the tray 715.
714 designate sending auxiliary rollers. The substratum 600 sent
into the tray 715 is attracted and immobilized tightly onto the
tray by the suction of the pump 716. The substratum 600 on the tray
715 is sent into an area where a first treatment step is carried
out. Numeral 704 designates a UV/O.sub.3 lump which provides the
pre-treatment of the base. When the base is transferred out of the
first step area by the sending motor 717, cellular screening
substances are applied with the micro-droplet discharging means
710. The base on which the cellular screening substances have been
applied is transferred immediately to an area where the third
immobilizing-treatment step is carried out, and the cellular
screening substances are immobilized on the base. Numeral 705
designates a UV irradiating lump. The base which has undergone
these three treating steps is transferred to the subsequent step
604 via the belt conveyor 720 and sending rollers 721.
[0074] However, the cellular screening apparatus according to the
claimed invention are not limited to these embodiments, if the
foregoing purposes are achieved, even though they are different
from these embodiments.
[0075] When it is not known what substance or which combination of
substances affect cellular functions of a certain cell type, at
least one function selected from the group consisting of adhesion,
proliferation, differentiation, survival, maintenance of
undifferentiated state and substance production, the cellular
screening substratum of the present invention can be used to
investigate such substances or combinations. Once such a substance
or substance combination was elucidated as a result of the
investigation, cells can be screened by using another cellular
screening substratum that is produced by disposing or disposing and
immobilizing such a substance or substances, or combinations on a
base by using, for example, microdroplet ejection means to screen
cells. Further, when a plurality of substances or combinations,
each of which affects different cellular function, are arranged or
immobilized after arrangement, one can obtain a cellular screening
substratum that enables screening of plural types of cells at the
same time.
EXAMPLES
[0076] The present invention is described in more detail by
Examples. These Examples are specific embodiments presented to
provide better understanding of the present invention, and not
intended to limit the present invention thereto in any way.
Example 1
[0077] (Cellular Screening Method of Evaluating Cell Growth
Factors)
[0078] A functional group was introduced in the following way, in
order to immobilize cellular screening substances on a base. A
solution of 50 mmol dicyclohexylcarbodiimide (DCC) in
tetrahydrofuran (THF) was added dropwise to a solution of 50 mmol
N-hydroxysuccinimide and 45 mmol 4-azidobenzenecarboxylic acid in
THF, and the reaction was carried out reacted at 4.degree. C. for
24 hours with stirring. The reaction product was dried under
reduced pressure, and then recrystallized and purified from an
isopropanol/diisopropanol solution. Subsequently, the reaction
product was dissolved in dimethylformamide, and to this solution
was added in small portions cellular screening substances dissolved
in an isotonic phosphate buffer solution (pH 7.0) The reaction was
carried out at 4.degree. C. for 48 hours to introduce azido groups
into the cellular screening substances.
[0079] In this Example, as the cellular screening substances were
used insulin, basic fibroblast growth factor (basic FGF), epidermal
growth factor (EGF), transforming growth factor-beta (TGF-.beta.),
and azide groups were introduced to each of these factors. Ink
cartridges were washed with purified water, and then filled with
isotonic phosphate buffer solutions (pH 7.0) containing each of the
cellular screening substances having the introduced functional
group and which were diluted in 50% methanol solution to a
concentration of 50 .mu.g/ml.
[0080] Next, as shown in FIG. 1, each of the cellular screening
substances 12 was discharged on a base 11 of a polyethylene
terephthalate (PET) film with an ink jet printer. Each cellular
screening substances 12 was discharged to respective areas for
immobilizing insulin 121, basic fibroblast growth factor 122,
epidermal growth factor 123, and transforming growth factor-.beta.
124, such that the substances were not overlapped to each other.
After drying droplets, a UV lump was used to irradiate the surface
of the base 11 with UV light to immobilize the cellular screening
substances 12. The base 11 was then washed with an isotonic
phosphate buffer solution (pH 7.0) to remove unreacted cellular
screening substances 12. A cellular screening substratum 1 was
manufactured in these procedures.
[0081] Cellular screening was carried out on this cellular
screening substratum 1.
[0082] The screening substratum which had been sterilized in
advance under a sterilizing lump was placed into a glass petri
dish, and as the culture medium was used a DMEM medium (Dulbecco's
Modified Eagle's minimum essential medium) supplemented with 10
.mu.g/ml transferin. On the screening substratum, vascular
endothelial cells were cultured under humidified air containing 5%
CO.sub.2 at 37.degree. C. for 24 hours. The culture medium also
contained .sup.3H-thimidine, and the amount of .sup.3H-thimidine
incorporated into the cells by growth was determined from the
amount of .sup.3H radiation in order to assess the degree of
proliferation.
[0083] When the substratum after culture was observed under a
microscope, growth of cells was observed in the insulin, basic-FGF,
and EGF immobilized areas, but not in the TGF-.beta. immobilized
area.
[0084] The growth density of cells was determined from the amount
of .sup.3H radiation. The insulin-immobilized area had a growth
density of 10000 cells/mm.sup.2, the basic-FGF immobilized area of
6000 cells/mm.sup.2, the EGF immobilized area of 8000
cells/mm.sup.2, and the TGF-.beta. immobilized area of 100
cells/mm.sup.2. These results demonstrated that insulin, basic-FGF,
and EGF have a growth activating effect on vascular endothelial
cells, whereas TGF-p does not have such an effect.
Example 2
[0085] (Cellular Screening Method of Evaluating Cell
Growth/Differentiation Factors)
[0086] Azide groups were introduced into insulin-like growth
factor-1 (IGF-1), basic-FGF, EGF, and TGF-.beta. by the
above-described method for introducing an immobilizing functional
group. As in Example 1, an ink jet printer was used to discharge
individual cellular screening substrata on a PET film to immobilize
them on a base. In this Example, the array or pattern was as shown
in FIG. 3. As shown in FIG. 3, on the substratum were provided an
isolated immobilized area 12a in which each of four cellular
screening substances was immobilized separately, and an interactive
immobilized area 12b in which two of the cellular screening
substances were close immobilized. Like this, one of the major
advantages of using droplet discharging means for applying cellular
screening substances to a base is that plural areas can be easily
formed on the same base varying the positional relationship between
the immobilization positions of two or more cellular screening
substances.
[0087] Chicken skeletal muscle cells were cultured on this
substratum using a DMEM medium supplemented with 10 .mu.g/ml
fibronectin, under similar conditions to those in Example 1. The
state of proliferation and differentiation was evaluated using an
Amersham Cell proliferation kit. This kit is for determining the
amount of synthesized DNA by a fluorescence antibody method using a
fluorescein-isothiocyanate (FITC) labeled antibody to
5-bromo-2'-deoxyuridine (BrdU) In addition, the growth density was
determined by staining the cells after culture. This procedure
involved treating the cultured cells with methanol for 30 minutes,
followed by drying them and staining the nuclei by an 10000 fold
dilution of Hoechst 33258 for 5 minutes. Excess staining solution
was washed away with an isotonic phosphate buffer solution. The
substratum was placed on a glass slide, and covered with a cover
glass after dropping glycerin. The substratum was observed under a
fluorescent microscope to count the number of stained nuclei. The
number of nuclei containing DNA labeled with BrdU was determined
quantitatively by a fluorescence quantification method. As a
consequent, in IGF-1 and EGF areas increased fluorescence and
close-packed nuclei were observed. It can be understood from these
results that the proliferation and differentiation of the cells
were promoted. In basic-FGF area, fluorescence increased but the
cell nuclei were dispersed, indicating that the proliferation was
activated, but the differentiation was not promoted. In TGF-.beta.
area, the increase in fluorescence was not substantial, so that it
is considered that the proliferation was not promoted. In the area
12b where combinations of two cellular screening substances were
arranged, it was observed that with the combination of IGF-1 and
EGF, each of which was active in proliferation and differentiation
in the area 12a, cell proliferation was suppressed with no increase
in the fluorescence amount. From these results, it has been found
that a combination of cellular screening substances that are active
singly may exert a suppressive effect.
Example 3
[0088] (Allergen Screening Method)
[0089] In this Example, constitution of a subject is evaluated
whether it is allergic or not by using a substratum with
immobilized possible allergenic substances and cells taken from the
subject and determining the amount of histamine, inflammation
causing substances produced by the cells during culture.
[0090] In this Example, a substratum as shown in FIG. 4 was
manufactured for assessing whether a subject is allergic to cedar
pollens, milk, house dusts, or wheat. The substratum was provided
with, in addition to an area 12a in which allergens were
immobilized singly, a two-substance immobilized area 12b, a
three-substance immobilized area 12c, and four-substance
immobilized area 12d in which two, three, and four allergens were
close immobilized respectively, so as to diagnose whether allergy
may be caused by the synergetic effect of two or more
allergens.
[0091] Cedar pollens, house dusts and wheat were fully grinded by a
homogenizer, and solutions containing each allergen were
centrifuged, and the solubilized fractions other than precipitation
were used for immobilization. Each allergen solution was then
diluted in 50% methanol to a concentration of 50 .mu.g/ml and
immobilized on a PET film with an ink jet printer by the method
described in Example 1. The immobilization pattern was as shown in
FIG. 4. The area not subjected to immobilization was coated with
bovine serum albumin to prevent non-specific adsorption.
[0092] Cells were collected from the blood of a subject to be
assessed, and blood components were separated by a density gradient
centrifugation to collect allergy-reactive cells.
[0093] The culture medium was a DMEM medium containing 10% fetal
bovine serum (FBS). An anti-histamine antibody (rabbit) was added
and the cells were cultured on the substratum.
[0094] The substratum was removed after culture and washed with an
isotonic phosphate buffer solution. After treating the substratum
with methanol for 30 minutes and drying, the histamine amount was
determined by an enzyme antibody method using a horseradish
peroxidase-conjugated anti-rabbit IgG antibody, measuring changes
in the absorbance of o-phenylenediamine. As a result, a high amount
of histamine was detected in the area where the extract of house
dusts was immobilized, therefore it is likely that this subject is
allergic to house dusts. Although this subject is considered
negative to the cedar pollens, milk, and wheat, it was found that a
high amount of histamine was detected in the area where milk and
wheat were immobilized in close vicinity, so that the subject may
develop allergy when both allergens are taken at the same time.
[0095] As described above, use of the substratum of the invention
enables determining the cause of allergy. In particular, it enables
diagnosis of an allergic reaction directed to plural allergens, as
in this Example.
Example 4
[0096] (Proliferation/Differentiation Screening Method with
Different Densities of Immobilized Substance)
[0097] In this Example, effects of EGF and TGF-.beta. on cell
growth of fibroblast NRK cells were studied. Azide groups were
introduced into these two cell growth factors EGF and TGF-.beta. in
advance according to the Example 1. The base was a PET film, and an
ink jet printer was used to discharged cell growth factors
respectively onto the substratum to dispose the factors varying the
density according to the positions on the base as shown in FIG. 5.
After drying, the base was irradiated with ultraviolet light to
immobilize the cell growth factors. The discharge amount was
controlled by setting the dot density of drawing data for
respective areas, which data were sent from the computer to the
printer. The figures indicated in FIG. 5 are relative dot densities
of each growth factor in a row or column direction, and each area
where a row and a column of respective densities cross, two growth
factors are immobilized at respective dot densities.
[0098] NRK cells were cultured on the substratum thus manufactured.
The culture medium was a DMEM medium supplemented with BrdU and 0.5
wt % FBS, and culture was carried out under humidified air with 5%
CO.sub.2 at 37.degree. C. for 48 hours.
[0099] After culture, evaluation was made using an Amersham Cell
proliferation kit. The results are shown in Table 1.
1 TABLE 1 Cell Density TGF-.beta.Density 10.sup.5 cells/mm.sup.2 0
4 20 100 EGF 0 14 42 34 30 Density 4 40 14 13 14 20 60 12 12 10 100
80 20 20 20
[0100] It is considered that the growth of NRK cells is promoted by
single EGF or TGF-.beta. but suppressed by combinations thereof.
From these effects of EGF and TGF-.beta. on cell growth of the NRK
cells, it is desirable to culture NRK cells in a culture medium
containing either of EGF and TGF-.beta. not both.
[0101] The cellular screening substrata of the present invention
have advantages that cellular screening substances can be
immobilized at desired positions by simple processing steps and
identifying substances contributing to at least one of adhesion,
proliferation and differentiation, survival, maintenance of
undifferentiated state, and apoptosis.
[0102] The methods of cellular screening of the present invention
make it possible to investigate effects on a cell exerted by
various substances on a single substratum, and additionally the
difference of the effects on different cells exerted by various
substances.
* * * * *