U.S. patent application number 16/208163 was filed with the patent office on 2019-06-06 for novel graft polymer, temperature-responsive substrate for cell culture using the same and production method therefor, as well as.
The applicant listed for this patent is Hideaki Sakai. Invention is credited to Masa-aki KAKIMOTO, Hideaki SAKAI, Yu SUDO.
Application Number | 20190169567 16/208163 |
Document ID | / |
Family ID | 51428425 |
Filed Date | 2019-06-06 |
United States Patent
Application |
20190169567 |
Kind Code |
A1 |
SAKAI; Hideaki ; et
al. |
June 6, 2019 |
NOVEL GRAFT POLYMER, TEMPERATURE-RESPONSIVE SUBSTRATE FOR CELL
CULTURE USING THE SAME AND PRODUCTION METHOD THEREFOR, AS WELL AS
LIQUID CHROMATOGRAPHIC CARRIER HAVING THE NOVEL GRAFT POLYMER
IMMOBILIZED THEREON AND LIQUID CHROMATOGRAPHIC METHOD USING THE
SAME
Abstract
By using a graft polymer comprising a dendritic polymer with a
styrene skeleton and a hydrophilic polymer grafted to a terminal
thereof, a temperature-responsive substrate for cell culture having
a temperature-responsive surface for cell culture that allows cells
to be cultured with high efficiency and which yet allows cultured
cells to be exfoliated in a short period of time and with high
efficiency by simply changing the temperature of the substrate
surface can be prepared conveniently. If this
temperature-responsive substrate for cell culture is used, cells
obtained from a variety of tissues can be cultured with high
efficiency. If this culture method is utilized, cultured cells can
be exfoliated intact in a short amount of time with high
efficiency. In addition, by using this graft polymer, a wide range
of peptides and proteins can also be separated by simply changing
the temperature of a chromatographic carrier. This allows for
convenient separation procedure and improves the efficiency of
separating operations. What is more, the stereoregularity of the
dendritic polymer per se may be utilized to enable separation of
solutes based on differences in their molecular structures.
Inventors: |
SAKAI; Hideaki;
(Kawasaki-shi, JP) ; KAKIMOTO; Masa-aki;
(Yokohama-shi, JP) ; SUDO; Yu; (Tokyo,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sakai; Hideaki |
Kawasaki-shi |
|
JP |
|
|
Family ID: |
51428425 |
Appl. No.: |
16/208163 |
Filed: |
December 3, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14771068 |
Nov 30, 2015 |
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PCT/JP2014/055178 |
Feb 28, 2014 |
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16208163 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08F 299/00 20130101;
C08F 293/005 20130101; C08L 101/04 20130101; B01D 15/3876 20130101;
C12M 23/20 20130101; C12N 2539/10 20130101; B01D 15/327 20130101;
B01J 20/3278 20130101; C08F 257/02 20130101; B01D 15/305 20130101;
B01J 20/286 20130101; C12N 2533/30 20130101; B01J 20/321 20130101;
B01J 20/264 20130101; C08L 2203/02 20130101; C12N 5/0068 20130101;
C08F 2438/03 20130101; C08L 51/003 20130101 |
International
Class: |
C12N 5/00 20060101
C12N005/00; B01J 20/32 20060101 B01J020/32; B01J 20/26 20060101
B01J020/26; B01J 20/286 20060101 B01J020/286; C08L 51/00 20060101
C08L051/00; C08L 101/04 20060101 C08L101/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 2013 |
JP |
2013-055630 |
Feb 28, 2013 |
JP |
2013-055631 |
Aug 27, 2013 |
JP |
2013-189831 |
Claims
1. A temperature-responsive substrate for cell culture comprising a
substrate coated with a dendritic polymer with a styrene skeleton
and a temperature-sensitive polymer grafted at a terminal
thereof.
2. The temperature-responsive substrate for cell culture according
to claim 1, which has electric charges at a terminal of the
dendritic polymer.
3. The temperature-responsive substrate for cell culture according
to claim 1, wherein the polymer having temperature response
comprises any one or more of a poly-N-substituted acrylamide
derivative, a poly-N-substituted methacrylamide derivative, a
copolymer thereof, polyvinyl methyl ether, and partially acetylated
polyvinyl alcohol, or a copolymer thereof with another monomer.
4. The temperature-responsive substrate for cell culture according
to claim 1, wherein the polymer having temperature response is
poly-N-isopropylacrylamide.
5. The temperature-responsive substrate for cell culture according
to claim 3, wherein the another monomer is a monomer having
electric charges and/or a hydrophobic monomer.
6. The temperature-responsive substrate for cell culture according
to claim 1, wherein any one or more of polyacrylamide,
poly-N,N-diethylacrylamide, poly-N,N-dimethylacrylamide, acrylate
having polyethylene oxide in side chains, and methacrylate having
polyethylene oxide in side chains are grafted to part of a terminal
of the dendritic polymer with a styrene skeleton.
7. The temperature-responsive substrate for cell culture according
to claim 1, which has a coating of 1.0 to 7.0 .mu.g/cm.sup.2 in
terms of the temperature-responsive polymer.
8. The temperature-responsive substrate for cell culture according
to claim 1, wherein the content of the temperature-responsive
polymer in the graft polymer ranges from 40 to 99.5 wt %.
9. The temperature-responsive substrate for cell culture according
to claim 1, wherein the molecular weight of the
temperature-responsive polymer in the graft polymer is 5000 or
more.
10. The temperature-responsive substrate for cell culture according
to claim 1, wherein a substrate is in the form of a particle, a
filament or a plate either individually or in combination of two or
more.
11. The temperature-responsive substrate for cell culture according
to claim 1, wherein a substrate is composed of polystyrene either
alone or combined with another material.
Description
[0001] This application is a continuation of application Ser. No.
14/771,068, which is the U.S. national phase of International
Application No. PCT/JP2014/055178 filed Feb. 28, 2014 which
designated the U.S. and claims priority to JP Patent Application
Nos. 2013-055630 and 2013-055631 filed Feb. 28, 2013, and
2013-189831 filed Aug. 27, 2013, the entire contents of each of
which are hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to a novel graft polymer that
is useful in biology, medicine and other fields, as well as a
temperature-responsive substrate for cell culture using the polymer
and a method for producing the substrate. The present invention
also relates to a carrier that can be prepared by a simple method
and which is suitable for use in liquid chromatography that is
carried out by controlling the interactions on a solid surface to
separate valuables such as pharmaceuticals, bio-related substances
(e.g. proteins, DNA, and glycolipids) and cells, as well as a
method of liquid chromatography using the carrier.
BACKGROUND ART
[0003] With recent remarkable advances in the animal cell culturing
technology, R&D activities targeting animal cells are spreading
in a wider range of fields. The way of using the target animal
cells has also changed; in the early stage of development, attempts
were made to commercialize animal cells per se or their products,
but now, it is becoming possible to design valuable pharmaceuticals
by analyzing cells or their outer surface proteins or perform
therapies with cells that have been taken out of a patient, then
either proliferated in number or enhanced in their functions, and
returned into the patient's body. Currently, the techniques of
culturing animal cells constitute one area that is attracting the
attention of many researchers.
[0004] One basic feature of animal cells including human cells is
that many of them are adhesion-dependent. In other words, animal
cells must be adhered to a certain place before they are cultured
ex vivo. Given this background, many researchers have so far
designed or invented substrate surfaces that would be more
favorable to cells but all proposals have been directed to the
phase during cell culture. When cultured adhesion-dependent cells
adhere to something else, they will produce adhesive proteins on
their own. Hence, in order to exfoliate the cells, the prior art
requires disrupting the adhesive proteins and an enzymatic
treatment is usually performed. In the process, outer surface
proteins that the cells have produced during culture and which are
inherent in the respective cells are also disrupted; in spite of
its seriousness, no effective means have been available to solve
the problem, with no particular studies made. Solving this problem
involved in the phase during cell recovery would be a requirement
which indeed must be satisfied before dramatic advances can be seen
in future R&D activities targeting animal cells.
[0005] High performance liquid chromatography (HPLC) has a wide
variety of combinations of the mobile-phase liquid and the
stationary phase, from which a suitable combination can be selected
depending upon the sample; hence, in recent years, HPLC is utilized
to separate and purify various substances. However, in the
conventionally used chromatography, interactions between the solute
in the mobile phase and the surface of the stationary phase are
driven not by changing the surface structure of the stationary
phase but mainly by changing the solvent in the mobile phase. For
example, in HPLC finding use in a lot of areas, normal-phase
columns in which a carrier such as silica gel is used as stationary
phase employ a mobile phase comprising an organic solvent such as
hexane, acetonitrile or chloroform, and reversed-phase columns for
separating highly water-soluble substances with a silica gel
derivative being used as a carrier employ an organic solvent such
as methanol or acetonitrile.
[0006] In ion-exchange chromatography which uses an anion exchanger
or a cation exchanger as stationary phase, the concentration or
type of the external ion is altered to separate substances. With
recent rapid advances in genetic engineering and like fields, use
of physiologically active peptides, proteins, DNA, etc. is desired
in various fields including pharmaceuticals, and their separation
and purification is an extremely important issue. In particular,
the need for techniques capable of separating and purifying
physiologically active substances without impairing their
activities is growing.
[0007] Unfortunately, however, organic solvents, acids, alkalis and
surfactants that are used in the conventional mobile phase not only
impair the activity of physiologically active substances, they are
also foreign substances, so improvements of the separation and
purification system are desired. It is also necessary to avoid the
environmental pollution by these substances and this is another
reason why a separation and purification system that does not use
them is required.
[0008] Given this background, various studies have heretofore been
made. Patent Document No. 1 discloses a novel cell culture method
which comprises providing a cell culture support having a substrate
surface coated with a polymer the upper or lower critical solution
temperature of which with respect to water is 0-80.degree. C.,
culturing cells on the support at a temperature either below the
upper critical solution temperature or above the lower critical
solution temperature, and subsequently adjusting the temperature
either to a point equal to or above the upper critical solution
temperature or to a point equal to below the lower critical
solution temperature, whereby the cultured cells are exfoliated
without enzymatic treatment.
[0009] Patent Document No. 2 discloses that using this
temperature-responsive cell culture substrate, skin cells are
cultured at a temperature either below the upper critical solution
temperature or above the lower critical solution temperature, and
subsequently adjusting the temperature either to a point equal to
or above the upper critical solution temperature or to a point
equal to below the lower critical solution temperature, whereby the
cultured skin cells are exfoliated while suffering low damage.
[0010] Patent Document No. 3 discloses a method of repairing outer
surface proteins on cultured cells using this
temperature-responsive cell culture substrate. By utilizing the
temperature-responsive cell culture substrate, it has become
possible to modify the conventional culture technology in various
novel ways.
By utilizing the temperature-responsive cell culture substrate, it
has now become possible to modify the conventional culture
technology in various novel ways. However, the technology here
mentioned involves coating the surfaces of chemically inactive
engineering plastics using high energy radiations such as an
electron beam and to this end, expensive equipment such as an
electron beam exposure unit is required and this has caused the
problem of an inevitable increase in the cost of the culture
substrate.
[0011] With a view to solving this problem, several techniques have
been developed to date. Representative among them are methods of
coating a substrate surface with synthesized polymers having the
particular molecular structures disclosed in Non-Patent Document
Nos. 1 and 2. But neither method is of such a technical level that
it is capable of culturing cells in a comparable way to the
conventional substrate for cell culture, that it enables the cells
to be exfoliated by simply changing the temperature in a comparable
way to the above-described temperature-responsive substrate for
cell culture which has been prepared using an electron beam, and
that the cultured cells are exfoliated as a cell sheet once they
have become confluent, and an improvement in their technical level
has been required. Considering this point, Patent Document No. 4
discloses that a substrate surface coated with a
temperature-responsive polymer joined to a block copolymer would
allow cultured cells to be exfoliated in a cell sheet form; as it
turned out, however, the substrate was simply coated with a uniform
layer of the temperature-responsive polymer and, hence, the ability
to exfoliate the cultured cells was limited.
[0012] Among the techniques of temperature-responsive
chromatography, the one disclosed in Patent Document No. 1 notably
provides a platform technology. Described in this document is a
technique which comprises providing a cell culture support
comprising a substrate surface coated with a polymer the upper or
lower critical solution temperature of which with respect to water
is 0-80.degree. C., culturing cells on the support at a temperature
either below the upper critical solution temperature or above the
lower critical solution temperature, and subsequently adjusting the
temperature either to a point equal to or above the upper critical
solution temperature or to a point equal to below the lower
critical solution temperature, whereby the cultured cells are
exfoliated. While this was the first case of using the
temperature-responsive polymer as a cell culture material in the
field of bio-medicine, the fact is that when cells are adhering to
the substrate surface, they will secrete adhesive proteins on their
own and use the secreted proteins as a linker for adhesion to the
substrate surface. Therefore, the phenomenon here contemplated of
cells exfoliating from the substrate surface implies that the
adhesive proteins secreted from the cells are also exfoliated from
the substrate surface. As a matter of fact, when cells obtained by
this technique are re-seeded or transplanted into a biological
tissue, the cells exfoliated from the substrate will efficiently
adhere to the substrate or tissue. This means that the exfoliated
cells retain the adhesive proteins as such after they were secreted
during culture. In other words, the technique of interest is
exactly the concept of the temperature-responsive chromatographic
technique as referred to in the present invention which involves
eliminating adsorbed proteins through temperature changes.
[0013] Patent Document No. 5 made a study about immobilization on
silica gel or polymer gel that is commonly used as chromatographic
carriers. However, even considering Examples, the document shows no
results (as separation charts) of solute separation in the case
where those carriers were actually used, and details as to what
substances could be separated using these carriers and what was the
specific issue to address were unknown.
[0014] In Patent Document No. 6, a temperature-responsive polymer
was immobilized on the surface of silica gel and cases are shown
where various steroids and even lymphocytes were actually separated
using the resulting carrier. It is definitely shown that the
various steroids and even lymphocytes were actually separated by
the characteristics of the temperature-responsive polymer
immobilized on the surface of the carrier silica gel. However,
considering the exemplary results of separation given in the
document, the number of theoretical stages at the separation needs
to be increased further and there has been a need to provide an
innovative technique that is improved over the prior art so greatly
as to solve this problem.
PRIOR ART LITERATURE
Patent Documents
[0015] Patent Document No. 1: JP H 02-211865 A [0016] Patent
Document No. 2: JP H 05-192138 A [0017] Patent Document No. 3: JP
2008-220354 A [0018] Patent Document No. 4: Japanese Patent
Application No. 2010-208506 [0019] Patent Document No. 5: JP H
05-133947 A [0020] Patent Document No. 6: JP H 07-318661 A
Non-Patent Documents
[0020] [0021] Non-Patent Document No. 1: Soft Matter, 5, 2937-2946
(2009) [0022] Non-Patent Document No. 2: Interface, 4, 1151-1157
(2007)
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0023] The present invention has been accomplished with the aim of
solving the aforementioned problems of the prior art. Briefly, the
present invention has as its primary objective providing a novel
graft polymer having better cell exfoliating functions based on a
concept totally different from the prior art.
[0024] Another object of the present invention is to provide a
novel temperature-responsive surface having better cell exfoliating
functions based on a concept totally different from the prior art
and a method of suing this surface.
[0025] Yet another object of the present invention is to provide a
novel liquid chromatographic carrier prepared on the basis of a
concept totally different from the prior art. A further object of
the present invention is to provide a liquid chromatographic method
using this carrier.
Means for Solving the Problems
[0026] In order to solve the aforementioned problems with cell
culture, the present inventors performed research and development
making studies from various angles and eventually came up with a
novel graft polymer. Surprisingly, they found that when cells were
cultured on a temperature-responsive substrate for cell culture
having a substrate surface coated with a graft polymer comprising a
dendritic polymer with a styrene skeleton and a
temperature-responsive polymer grafted at a terminal thereof, the
cultured cells could be efficiently exfoliated by simply cooling
the substrate for a short period of time. From the early stage of
their research activities, the present inventors focused on the
special stereoregularity and morphology on substrate surface of the
graft copolymer in which the dendritic polymer with a styrene
skeleton was more compact in structure than the linear styrene and
of such a structure that the temperature-responsive polymer was
concentrated at a terminal of the dendritic polymer, and they were
in great hopes that the substrate for cell culture formed from this
graft polymer would have higher performance than the prior art
temperature-responsive substrate for cell culture. It was also
found that the graft polymer that comprised the dendritic polymer
with a styrene skeleton and the temperature-responsive polymer
grafted at a terminal thereof and which was applied as a coating to
the surface of the substrate of the present invention was insoluble
in water and, hence, would neither dissolve out into a culture
medium during cell culture nor contaminate the cultured cells
during their recovery. It was also found that because of these
advantages, the temperature-responsive substrate for cell culture
of the present invention can be used over and over again. The
present invention has been accomplished on the basis of these
findings.
[0027] In order to solve the aforementioned problems with
chromatographic carriers, the present inventors also performed
research and development making studies from various angles. As a
result and surprisingly enough, they found that the dendritic
polymer with a styrene skeleton could be conveniently immobilized
in a thin layer on the surface of a resin-based chromatographic
carrier and that when the temperature-responsive polymer was
grafted to this dendritic polymer, the carrier's surface displayed
such a phenomenon that its chromatographic resolution would
abruptly change at a certain temperature. At the early stage of
their research activities, the present inventors focused on the
stereoregularity of the dendritic polymer and the functional groups
occurring at high density on the outside of the molecular chain and
speculated that if this dendritic polymer was immobilized on the
surface of a chromatographic carrier, the functional groups could
be provided at high density on the carrier's surface and that if
the above-described temperature-responsive polymer was joined to
the dendritic polymer, the product would have higher performance
than the prior art temperature-responsive chromatographic carrier.
The technique offered by the present invention is totally
unexpected from the prior art and if combined with the
stereoregularity of the dendritic polymer structure per se, it is
expected to evolve to a novel chromatographic system that has never
existed in the prior art. The present invention has been
accomplished on the basis of these findings.
[0028] Thus, the present invention provides a graft polymer as a
novel material comprising a dendritic polymer with a styrene
skeleton and a temperature-responsive polymer grafted at a terminal
thereof.
[0029] Further, the present invention provides a
temperature-responsive substrate for cell culture that has a
substrate surface coated with a graft polymer comprising a
dendritic polymer with a styrene skeleton and a
temperature-responsive polymer grafted at a terminal thereof. The
present invention also provides a method for producing the
temperature-responsive substrate for cell culture.
[0030] Additionally, the present invention provides a liquid
chromatographic carrier on which is immobilized a graft polymer
comprising a dendritic polymer with a styrene skeleton and a
polymer of different composition grafted at a terminal thereof. The
present invention also provides a liquid chromatographic method
using the liquid chromatographic carrier.
[0031] The present invention is briefly described below.
[1] A novel graft polymer comprising a dendritic polymer with a
styrene skeleton and a hydrophilic polymer grafted at a terminal
thereof. [2] The novel graft polymer as recited in [1], which has
electric charges at a terminal of the dendritic polymer. [3] The
novel graft polymer as recited in any one of [1] and [2], wherein
the hydrophilic polymer is one having temperature response. [4] The
novel graft polymer as recited in [3], wherein the polymer having
temperature response comprises any one or more of a
poly-N-substituted acrylamide derivative, a poly-N-substituted
methacrylamide derivative, a copolymer thereof, polyvinyl methyl
ether, and partially acetylated polyvinyl alcohol, or a copolymer
thereof with another monomer. [5] The novel graft polymer as
recited in [4], wherein the polymer having temperature response is
poly-N-isopropylacrylamide. [6] The novel graft polymer as recited
in [4], wherein the another monomer is a monomer having electric
charges and/or a hydrophobic monomer. [7] The novel graft polymer
as recited in any one of [1] and [2], wherein the hydrophilic
polymer comprises any one or more of polyacrylamide,
poly-N,N-diethylacrylamide, poly-N,N-dimethylacrylamide, acrylate
having polyethylene oxide in side chains, and methacrylate having
polyethylene oxide in side chains. [8] The novel graft polymer as
recited in any one of [1] and [2], wherein the hydrophilic polymer
is a mixture of the polymers as recited in any one of [3] to [7].
[9] A temperature-responsive substrate for cell culture comprising
a substrate coated with a dendritic polymer with a styrene skeleton
and a temperature-sensitive polymer grafted at a terminal thereof.
[10] The temperature-responsive substrate for cell culture as
recited in [8], which has electric charges at a terminal of the
dendritic polymer. [11] The temperature-responsive substrate for
cell culture as recited in any one of [9] and [10], wherein the
polymer having temperature response comprises any one or more of a
poly-N-substituted acrylamide derivative, a poly-N-substituted
methacrylamide derivative, a copolymer thereof, polyvinyl methyl
ether, and partially acetylated polyvinyl alcohol, or a copolymer
thereof with another monomer. [12] The temperature-responsive
substrate for cell culture as recited in any one of [9] to [11],
wherein the polymer having temperature response is
poly-N-isopropylacrylamide. [13] The temperature-responsive
substrate for cell culture as recited in [11], wherein the another
monomer is a monomer having electric charges and/or a hydrophobic
monomer. [14] The temperature-responsive substrate for cell culture
as recited in any one of [9] to [13], wherein any one or more of
polyacrylamide, poly-N,N-diethylacrylamide,
poly-N,N-dimethylacrylamide, acrylate having polyethylene oxide in
side chains, and methacrylate having polyethylene oxide in side
chains are grafted to part of a terminal of the dendritic polymer
with a styrene skeleton. [15] The temperature-responsive substrate
for cell culture as recited in any one of [9] to [14], which has a
coating of 1.0 to 7.0 .mu.g/cm.sup.2 in terms of the
temperature-responsive polymer. [16] The temperature-responsive
substrate for cell culture as recited in any one of [9] to [15],
wherein the content of the temperature-responsive polymer in the
graft polymer ranges from 40 to 99.5 wt %. [17] The
temperature-responsive substrate for cell culture as recited in any
one of [9] to [16], wherein the molecular weight of the
temperature-responsive polymer in the graft polymer is 5000 or
more. [18] The temperature-responsive substrate for cell culture as
recited in any one of [9] to [17], wherein a substrate is in the
form of a particle, a filament or a plate either individually or in
combination of two or more. [19] The temperature-responsive
substrate for cell culture as recited in any one of [9] to [18],
wherein a substrate is composed of polystyrene either alone or
combined with another material. [20] A method for producing a
temperature-responsive substrate for cell culture by dissolving or
dispersing a graft polymer in an organic solvent, applying a
solution of the graft polymer uniformly onto a substrate surface,
and drying the same. [21] The method for producing a
temperature-responsive substrate for cell culture as recited in
[20], wherein the organic solvent is a liquid mixture of
tetrahydrofuran and methanol. [22] The method for producing a
temperature-responsive substrate for cell culture as recited in
[21], wherein the mixing ratio of tetrahydrofuran and methanol in
the mixed solvent is 1:4. [23] A liquid chromatographic carrier
comprising a carrier surface on which a graft polymer is
immobilized, wherein the graft polymer comprises a dendritic
polymer with a styrene skeleton and a polymer of a different
composition joined to a terminal thereof. [24] The liquid
chromatographic carrier as recited in [23], wherein the polymer
grafted to the dendritic polymer with a styrene skeleton comprises
a polymer having temperature response, and any one or two of a
hydrophilic polymer, and a hydrophobic polymer. [25] The liquid
chromatographic carrier as recited in [24], wherein the polymer
having temperature response comprises any one or more of a
poly-N-substituted acrylamide derivative, a poly-N-substituted
methacrylamide derivative, a copolymer thereof, polyvinyl methyl
ether, and partially acetylated polyvinyl alcohol, or a copolymer
thereof with another monomer. [26] The liquid chromatographic
carrier as recited in [25], wherein the polymer having temperature
response is poly-N-isopropylacrylamide. [27] The
temperature-responsive surface as recited in any one of [25] and
[26], wherein the molecular weight of the polymer having
temperature response is 5000 or more. [28] The
temperature-responsive surface as recited in any one of [25] to
[27], wherein the content of the polymer having temperature
response in the graft polymer ranges from 40 to 99.5 wt %. [29] The
liquid chromatographic carrier as recited in [24], wherein the
hydrophilic polymer comprises any one or more of polyacrylamide,
poly-N,N-diethylacrylamide, poly-N,N-dimethylacrylamide, acrylate
having polyethylene oxide in side chains, and methacrylate having
polyethylene oxide in side chains. [30] The liquid chromatographic
carrier as recited in any one of [23] to [29] which has electric
charges at a terminal of the dendritic polymer. [31] The liquid
chromatographic carrier as recited in any one of [23] to [30] which
has electric charges on the polymer grafted to the dendritic
polymer with a styrene skeleton. [32] The liquid chromatographic
carrier as recited in any one of [23] to [31], wherein the graft
polymer is immobilized on the surface of the carrier at a dose of
1.0 to 7.0 .mu.g/cm.sup.2. [33] The liquid chromatographic carrier
as recited in any one of [23] to [32], wherein a carrier is
composed of polystyrene either alone or combined with another
material. [34] The liquid chromatographic carrier as recited in any
one of [23] to [33], wherein the carrier is in the form of a
particle, a filament or a plate either individually or in
combination of two or more. [35] A method for producing a liquid
chromatographic carrier by dissolving or dispersing a graft polymer
in an organic solvent, applying a solution of the polymer uniformly
onto a substrate surface, and drying the same. [36] The method for
producing a liquid chromatographic carrier as recited in [35],
wherein the organic solvent is a liquid mixture of tetrahydrofuran
and methanol. [37] The method for producing a liquid
chromatographic carrier as recited in [36], wherein the mixing
ratio of tetrahydrofuran and methanol in the mixed solvent is 1:4.
[38] A liquid chromatographic method using the liquid
chromatographic carrier as recited in any one of [23] to [34]. [39]
The liquid chromatographic method as recited in [38] which
comprises separating a solute under specified temperature
conditions. [40] The liquid chromatographic method as recited in
[38] which comprises separating a solute while varying the
temperature such that it crosses the level at which the properties
of the carrier surface will change. [41] The liquid chromatographic
method as recited in [38], wherein the temperature variation is
either in an intermittent or continuous way or in both intermittent
and continuous ways. [42] The liquid chromatographic method as
recited in [38] which comprises adsorbing the solute on the liquid
chromatographic carrier and subsequently varying the temperature to
change the properties of the carrier surface so that the adsorbed
solute becomes free. [43] The liquid chromatographic method as
recited in [38] which comprises separating the solute with two or
more types of the liquid chromatographic carrier packed in the same
column by varying the temperature such that it crosses the level at
which the properties of the carrier surface will change. [44] The
liquid chromatographic method as recited in [38] which comprises
separating the solute with the liquid chromatographic carrier in a
column the inlet and outlet temperatures of which are so set that
the temperature level at which the properties of the carrier
surface will change is in between said inlet and outlet
temperatures and that a temperature gradient is provided within the
column from the inlet to the outlet end. [45] The liquid
chromatographic method as recited in any one of [38] to [44],
wherein the mobile phase is an aqueous system. [46] The liquid
chromatographic method as recited in any one of [38] to [45] which
is for separating pharmaceuticals, metabolites thereof,
agrochemicals, peptides or proteins.
Effects of the Invention
[0032] By using substrates coated on their surface with the novel
graft polymer to be described herein, cells can be cultured
efficiently and, what is more, the cultured cells can be exfoliated
efficiently within a short period of time by simply changing the
temperature of the substrate surface. And substrates having such
functional surface can be conveniently prepared in accordance with
the production method of the present invention.
[0033] Additionally, a novel separation system is offered by the
liquid chromatographic carrier described herein. Given this system,
peptides and proteins can be separated over a wide range. And the
stereoregularity of the dendritic polymer itself may be utilized to
enable solutes to be separated depending on the differences in
their molecular structures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is a diagram showing the synthesis pathway of a
dendritic polymer with a styrene skeleton in Example 1.
[0035] FIG. 2 is a diagram showing the synthesis pathway of a graft
polymer comprising the dendritic polymer with a styrene skeleton
and a temperature-responsive polymer grafted at a terminal thereof
according to Example 1.
[0036] FIG. 3 is a table showing the conditions for synthesis of
graft polymers each comprising the dendritic polymer with a styrene
skeleton and a temperature-responsive polymer grafted at a terminal
thereof according to Example 1.
[0037] FIG. 4 is a table showing the conditions for synthesis of
graft polymers each comprising the dendritic polymer with a styrene
skeleton and a temperature-responsive polymer grafted at a terminal
thereof according to Example 1.
[0038] FIG. 5 is a table showing the conditions for synthesis of
graft polymers each comprising the dendritic polymer with a styrene
skeleton and a temperature-responsive polymer grafted at a terminal
thereof according to Example 1.
[0039] FIG. 6 is a table showing the results of synthesis of graft
polymers each comprising the dendritic polymer with a styrene
skeleton and a temperature-responsive polymer grafted at a terminal
thereof according to Example 1.
[0040] FIG. 7 is a chart depicting the result of synthesizing the
dendritic polymer with a styrene skeleton according to Example
1.
[0041] FIG. 8 is a chart depicting the result of synthesizing a
graft polymer comprising the dendritic polymer with a styrene
skeleton and a temperature-responsive polymer grafted at a terminal
thereof according to Example 1.
[0042] FIG. 9 is a chart depicting the result of synthesizing a
graft polymer comprising the dendritic polymer with a styrene
skeleton according to Example 1 and a temperature-responsive
polymer grafted at a terminal thereof.
[0043] FIG. 10 is a chart depicting the result of synthesizing a
graft polymer comprising the dendritic polymer with a styrene
skeleton and a temperature-responsive polymer grafted at a terminal
thereof according to Example 1.
[0044] FIG. 11 shows in photo the result of culturing cells on a
temperature-responsive surface prepared in Example 3.
[0045] FIG. 12 shows in photo the result of culturing cells on
another temperature-responsive surface prepared in Example 3.
[0046] FIG. 13 shows in photo the result of culturing cells on yet
another temperature-responsive surface prepared in Example 3.
[0047] FIG. 14 is a set of charts depicting the results of steroid
separation in Example 5.
BEST MODES FOR CARRYING OUT THE INVENTION
[0048] The present invention provides a graft polymer comprising a
dendritic polymer with a styrene skeleton and a
temperature-responsive polymer grafted at a terminal thereof as
well as a temperature-responsive substrate for cell culture
comprising a substrate surface coated with the graft polymer. The
dendritic polymer with a styrene skeleton in the graft polymer
coating on the substrate surface will effectively prevent the
coating from becoming free from the substrate surface not only
during cell culture but also when the cultured cells are exfoliated
through temperature change which is the major feature of the
present invention. The dendritic polymer as described herein is not
particularly limited as long as it has a styrene skeleton. The
method of its production also is not particularly limited and it
may, for example, be obtained by atom transfer radical
polymerization (ATRP) which is commonly performed in chlorobenzene
in the presence of copper chloride. In the present invention, in
order to extend a temperature-responsive polymer from a terminal of
the dendritic polymer with a styrene skeleton, styrene derivatives
having functional groups as exemplified by chloromethylstyrene,
bromomethylstyrene or other halogenated methylstyrene species need
be used either alone or in combinations of two or more species. In
that case, the mixing ratio of styrene derivatives having
functional groups in all monomers that compose the dendritic
polymer typically ranges from 5% (inclusive) to 90% (inclusive),
preferably from 10% to 80% (i.e., not less than 10% but not more
than 80%), more preferably from 15% to 70% (i.e., not less than 15%
but not more than 70%), and most preferably from 20% to 60% (i.e.,
not less than 20% but not more than 60%). If the mixing ratio of
styrene derivatives having functional groups is less than 5%, the
efficiency of introducing the temperature-responsive polymer chain
from a terminal becomes so low that the percent introduction of
temperature-responsive polymer that is desired for the present
invention is not attained and the product is not preferred as the
graft polymer of the present invention. What is more, the resulting
graft polymer is so similar in properties to the polystyrene
substrate for its coating that organic solvents that dissolve the
graft polymer will also dissolve the substrate surface and no
suitable solvents are available for coating the substrate surface
with the graft polymer, which is again unfavorable for the purposes
of the present invention. On the other hand, if the mixing ratio of
styrene derivatives having functional groups is more than 90%, the
efficiency of introducing the temperature-responsive polymer chain
from a terminal is improved, producing a graft polymer in which the
properties of the temperature-responsive polymer predominate; as a
result, the graft polymer may become so highly soluble in water
that it is likely to dissolve out into the culture medium during
cell culture and may even contaminate the cultured cells in the
process of recovery; this graft polymer is also unsuitable for use
in the present invention. The molecular weight of the dendritic
polymer also is not particularly limited but considering its
relative proportion with respect to the temperature-responsive
polymer to which it is joined, the molecular weight of interest is
recommended to range from 2000 to 20000 (i.e., not less than 2000
but not more than 20000), preferably from 2500 to 15000 (i.e., not
less than 2500 but not more than 15000), more preferably from 3000
to 10000 (i.e., not less than 3000 but not more than 10000), and
most preferably from 4000 to 8000 (i.e., not less than 4000 but not
more than 8000). If the molecular weight of the dendritic polymer
is smaller than 2000, the relative proportion of the
temperature-responsive polymer chain is increased, producing a
graft polymer in which the properties of the temperature-responsive
polymer predominate; as a result, the graft polymer will become so
highly soluble in water that it is likely to dissolve out into the
culture medium during cell culture and may even contaminate the
cultured cells in the process of recovery, which is not preferred
as the graft polymer of the present invention. If, on the other
hand, the molecular weight of the dendritic polymer is greater than
20000, the percent introduction of temperature-responsive polymer
that is desired for the present invention is not attained and the
product is not preferred as the graft polymer of the present
invention. What is more, the resulting graft polymer is so similar
in properties to the polystyrene substrate for its coating that
organic solvents that dissolve the graft polymer will also dissolve
the substrate surface and no suitable solvents are available for
coating the substrate surface with the graft polymer, which is
again unfavorable for the purposes of the present invention. If
desired, in the case of the present invention, positive or negative
charges as on hydroxyl, carboxyl, amino, carbonyl, aldehyde or
sulfonic groups may be added to a terminal of the dendritic polymer
in the usual manner; alternatively, positive or negative charges as
on hydroxyl, carboxyl, amino, carbonyl, aldehyde or sulfonic groups
may be left in the dendritic polymer moiety of the graft polymer as
the final form of the compound of the present invention which
comprises the dendritic polymer with a styrene skeleton and the
temperature-responsive polymer joined to a terminal thereof.
[0049] The present invention also provides a liquid chromatographic
carrier characterized in that a graft polymer is immobilized
wherein the graft polymer comprises a dendritic polymer with a
styrene skeleton and a polymer of a different composition joined to
a terminal thereof, as well as a liquid chromatographic method
using the liquid chromatographic carrier. The present inventors
conducted various studies with a view to meeting the
above-mentioned demands for liquid chromatographic carriers; as a
result, they developed a technique in which separation/purification
could be achieved by changing the interactions between the solute
and the surface of the stationary phase through a change in an
external condition such as temperature on the surface structure of
the stationary phase, rather than through a change of the mobile
phase. The present invention has been accomplished on the basis of
this technique and aims to provide a chromatographic method that
involves changing an external condition whereby the surface
properties of the stationary phase are changed reversibly so that
separation/purification is possible using a single aqueous mobile
phase, as well as to provide a packing agent suitable for use as
the stationary phase in the chromatographic method. In essence, the
present invention provides a chromatographic method characterized
by performing solute separation with such a packing agent that if,
for example, the graft polymer comprising the dendritic polymer and
a temperature-responsive polymer joined thereto is immobilized on a
carrier surface serving as the stationary phase, the properties of
the surface of the stationary phase will be varied with temperature
while the mobile phase is fixed to an aqueous system. The present
invention further provides a temperature-responsive chromatographic
method using said packing agent. Stated briefly, the use of the
present invention enables bio-elements such as peptides, proteins
or cells to be separated by bringing the outside temperature to a
level equal to or above a critical point. In the process, no
chemicals including organic solvents, acids, alkalis, surfactants,
etc. are used, so the present invention eliminates the possibility
that such chemicals will become foreign substances and can
additionally be utilized in separating proteins, cells and other
bio-elements in the same manner as they are analyzed with their
functions kept intact.
[0050] If the conventional chromatographic method is applied, with
a single type of mobile phase, to separate and analyze samples
containing a variety of compounds in admixture, in particular, a
plurality of samples having greatly different polarities,
separation is difficult and requires a considerable amount of time
to be complete. Therefore, to handle such samples, the amount and
type of the organic solvent are changed over time either
continuously (solvent gradient method) or in stages (step gradient
method). In the temperature gradient method or step gradient method
according to the present invention, the same level of separation
can be achieved by changing the column temperature either
continuously or in stages using a single mobile phase rather than
organic solvents. By adopting this approach, contamination by the
aforementioned foreign substances can be prevented while ensuring
that proteins, cells and other bio-elements can be separated with
their functions kept intact and, what is more, the desired
components can be separated within a short period of time through
temperature-mediated control.
[0051] The present invention is described below in a more specific
way. The present invention provides a liquid chromatographic
carrier on which a graft polymer is immobilized wherein the graft
polymer comprises a dendritic polymer with a styrene skeleton and a
polymer of a different composition joined to a terminal thereof.
And even if only the dendritic polymer is immobilized in a thin
layer on the carrier surface, the latter will develop a
temperature-responsive property. Although the reason for this
phenomenon is not clear as yet, the functions of the dendritic
polymer per se presumably changed by a considerable degree as a
result of the polymer having been immobilized in a thin layer on
the carrier surface, leading to the binding of its molecular chain.
In the present invention, factors selected from among the
hydrophilicity/hydrophobicity of chromatographic carrier, the
degree by which hydrophobic groups in the molecular chain of the
dendritic polymer are exposed on the carrier surface, the
fluctuation of the molecular chain, the molecule recognizing
ability of the molecular chain, exclusion limit, glass transition
point, and so forth may have acted either individually or in
superposition but it should be understood that this reason will by
no means limit the technology of the present invention.
[0052] Examples of the temperature-responsive polymer include
polymers having a lower critical solution temperature (LCST) and
polymers having an upper critical solution temperature (UCST), and
these polymers may be homopolymers, copolymers or mixtures thereof.
Examples of such polymers include those disclosed in JP H 06-104061
B. Specifically, these may be obtained by homopolymerization or
copolymerization of the monomers named below. Applicable monomers
include, for example, (meth)acrylamide compounds, N-(or
N,N-di)alkyl-substituted (meth)acrylamide derivatives, or vinyl
ether derivatives, and partially acetylated polyvinyl alcohol. In
the case of copolymers, any two or more of these monomers may be
used. In this case, considering that separation is performed at the
range of 5.degree. C. to 50.degree. C. since the substances to be
separated are bio-substances, exemplary temperature-responsive
polymers include poly-N-n-propylacrylamide (21.degree. C. as the
lower critical solution temperature of the homopoloymer),
poly-N-n-propylmethacrylamide (27.degree. C. as defined above),
poly-N-isopropylacrylamide (32.degree. C. as defined above),
poly-N-isopropylmethacrylamide (43.degree. C. as defined above),
poly-N-cyclopropylacrylamide (45.degree. C. as defined above),
poly-N-ethoxyethylacrylamide (ca. 35.degree. C. as defined above),
poly-N-ethoxyethylmethacrylamide (ca. 45.degree. C. as defined
above), poly-N-tetrahydrofurfurylacrylamide (ca. 28.degree. C. as
defined above), poly-N-tetrahydrofurfurylmethacrylamide (ca.
35.degree. C. as defined above), poly-N,N-ethylmethylacrylamide
(ca. 56.degree. C. as defined above), poly-N,N-diethylacrylamide
(32.degree. C. as defined above), etc. What is more,
copolymerization with monomers other than those mentioned above,
grafting or copolymerization between polymers, or mixtures of
polymers or copolymers may be employed. If desired, polymers may be
crosslinked to such an extent that their inherent properties will
not be impaired. Monomers that may be used in the process are not
particularly limited and exemplary hydrophobic monomers include
alkyl acrylates such as n-butyl acrylate and t-butyl acrylate, and
alkyl methacrylates such as n-butyl methacrylate, t-butyl
methacrylate, and methyl methacrylate. Additionally, exemplary
ionic monomers having charge-generating functional groups include
the following: building blocks of polymers having an amino group,
such as dialkylaminoalkyl(meth)acrylamide, dialkylaminoalkyl
(meth)acrylate, aminoalkyl (meth)acrylate, aminostyrene,
aminoalkylstyrene, aminoalkyl(meth)acrylamide,
alkyloxyalkyltrimethyl ammonioum salt, and
3-acrylamidopropoyltrimethyl ammonium chloride as a
(meth)acrylamidealkyltrimethyl ammonium salt; building blocks of
polymers having a carboxyl group, such as acrylic acid and
methacrylic acid; and building blocks of polymers having a sulfonic
group, such as (meth)acrylamidoalkylsulfonic acid; it should,
however, be noted that these are not the sole examples that can be
used in the present invention.
[0053] In the present invention, the dendritic polymer may have
another polymer grafted thereto by such a degree that the
temperature response desired in the present invention will not be
impaired. Applicable polymers are not particularly limited and
hydrophilic polymers may be mentioned. The hydrophilic polymers to
be used in the present invention may be either homopolymers or
copolymers. Examples include, but are not particularly limited to,
polyacrylamide, poly-N,N-diethylacrylamide,
poly-N,N-dimethylacrylamide, polyethylene oxide, acrylates having
polyethylene oxide in side chains, methacrylates having
polyethylene oxide in side chains, polyacrylic acid and salts
thereof, and hydrous polymers such as poly(hydroxyethyl
methacrylate), poly(hydroxyethyl acrylate), polyvinyl alcohol,
polyvinylpyrrolidone, cellulose, and carboxymethyl cellulose.
[0054] In the present invention, the foregoing procedure yields a
graft polymer comprising the dendritic polymer with a styrene
skeleton and the temperature-responsive polymer grafted to a
terminal thereof. The content of the temperature-responsive polymer
in the graft polymer typically ranges from 40.0 to 99.5 wt % (i.e.,
not less than 40.0 wt % but not more than 99.5 wt %), preferably
from 50 to 99 wt % (i.e., not less than 50 wt % but not more than
99 wt %), more preferably from 70 to 98 wt % (i.e., not less than
70 wt % but not more than 98 wt %), and most preferably from 85 to
97 wt % (i.e., not less than 85 wt % but not more than 97 wt %).
Below 40.0 wt %, cultured cells on the graft polymer are difficult
to exfoliate even if the temperature is changed and the operational
efficiency will drop markedly, which is unfavorable for the
purposes of the present invention. What is more, as mentioned
earlier, the resulting graft polymer is so similar in properties to
the polystyrene substrate for coating that organic solvents that
dissolve the graft polymer will also dissolve the substrate surface
and no suitable solvents are available for coating the substrate
surface with the graft polymer, which is again unfavorable for the
purposes of the present invention. On the other hand, above 99.5 wt
%, the graft polymer of interest will become so highly soluble in
water that it is likely to dissolve out into the culture medium
during cell culture and may even contaminate the cultured cells in
the process of recovery; this graft polymer is also unsuitable for
use in the present invention.
[0055] While the temperature-responsive polymer which composes the
graft polymer in the present invention can be used if its molecular
weight is 3000 and more, the recommended molecular weight is at
least 5000, preferably at least 10000, more preferably at least
17000, and most preferably at least 20000. If the molecular weight
is smaller than 3000, cultured cells on that polymer are difficult
to exfoliate even if the temperature is changed and the operational
efficiency will drop markedly, which is unfavorable for the
purposes of the present invention.
[0056] The graft polymer in the present invention is applied as a
coating that contains the temperature-sensitive polymer in an
amount ranging from 1.0 to 7.0 .mu.g/cm.sup.2 (i.e., not less than
1.0 .mu.g/cm.sup.2 but not more than 7.0 .mu.g/cm.sup.2),
preferably from 2.0 to 5.0 .mu.g/cm.sup.2 (i.e., not less than 2.0
.mu.g/cm.sup.2 but not more than 5.0 .mu.g/cm.sup.2), more
preferably from 2.5 to 4.5 .mu.g/cm.sup.2 (i.e., not less than 2.5
.mu.g/cm.sup.2 but not more than 4.5 .mu.g/cm.sup.2), and most
preferably from 3.0 to 3.5 .mu.g/cm.sup.2 (i.e., not less than 3.0
.mu.g/cm.sup.2 but not more than 3.5 .mu.g/cm.sup.2). If the
coating weight is smaller than 1.0 .mu.g/cm.sup.2, cultured cells
on the graft polymer are difficult to exfoliate even if the
temperature is changed and the operational efficiency will drop
markedly, which is unfavorable for the purposes of the present
invention. On the other hand, if the coating weight is greater than
7.0 .mu.g/cm.sup.2, cells will not readily adhere to the coated
area, making it difficult to ensure adequate adhesion of the cells;
the resulting substrate for cell culture is by no means preferred
for use in the present invention.
[0057] In the case of the present invention, the graft polymer
comprising the dendritic polymer with a styrene skeleton and the
temperature-responsive grafted to a terminal thereof needs to be
applied to a substrate surface in only the required amount. Since
the graft polymer of the present is water-insoluble, it does not
need to be washed after the coating step; hence, the amount of the
temperature-responsive polymer in the applied graft polymer will
directly contribute to the temperature-responsive polymer on the
substrate surface. If necessary, the coating weight of the
temperature-responsive polymer may be measured in the usual manner
as by FT-IR-ATR, elemental analysis or ESCA, and any one of these
methods may be employed.
[0058] The graft polymer of the present invention is such that the
dendritic polymer with a styrene skeleton which is a
water-insoluble polymer and the temperature-responsive polymer
which has affinity for water are joined together. Therefore, if
this graft polymer is coated on a substrate surface and dried, a
fine phase-separated structure is anticipated to form on the
substrate surface. While the phase-separated structure is not
particularly limited in morphology, size, etc., any presence of the
phase-separated structure on the substrate surface to which cells
are adhering will enable suppression of their degeneration, which
is favorable for the purposes of the present invention.
[0059] It should also be mentioned that in the case of the present
invention, the dithioester-based functional group which is part of
the structure of the RAFT agent will remain at a terminal of the
resulting polymer. This is a characteristic phenomenon in RAFT
polymerization and after the polymerization reaction ends,
additional polymerization reaction can be initiated from that
terminal. In the process, the dithioester-based functional group
present at a terminal of the temperature-responsive polymer is
readily replaced by a thiol group by adding a suitable compound
such as 2-ethanolamine. This reaction need not be carried out under
any special conditions and it is not only convenient but is a rapid
reaction that proceeds in a short period of time. As a consequence,
one can obtain a polymer chain having the highly reactive thiol
group and, hence, the polymer chain can be selectively and
efficiently modified at a terminal with functional molecules having
functional groups such as a maleimide group or a thio group.
Accordingly, the surface of the temperature-responsive substrate
for cell culture and the surface of the temperature-responsive
liquid chromatographic carrier, both according to the present
invention, can be provided with new functional features. In the
process, the types of functional groups are not particularly
limited and may include, for example, a hydroxyl group, a carboxyl
group, an amino group, a carbonyl group, an aldehyde group, and a
sulfonic group. If desired, peptides or proteins that will promote
cell adhesion may be immobilized at a terminal of the polymer
chain. In this connection, the lower critical solution temperature
(LCST) of poly-N-isopropylacrylamide is variable depending on the
hydrophilicity or hydrophobicity of the terminal functional group,
the approach of introducing functional groups at a terminal of the
polymer chain as in the present invention has a potential to
provide a new technique for controlling the temperature response of
the substrate surface from an unconventional viewpoint.
[0060] In the present invention, the above-mentioned variety of
polymers is immobilized at a terminal of the dendritic polymer
having a styrene skeleton and the method of immobilization is not
particularly limited. An exemplary method comprises converting the
halogen terminal of the above-mentioned halogenated methylstyrene
species to an azide form, then modifying the terminal with an
initiator (CTA) of reversible addition-fragmentation chain transfer
(RAFT) polymerization by click chemistry, and allowing a variety of
monomers to grow from the initiator. The initiator used in the
process is not particularly limited and examples include
2,2'-azobis(isobutyronitrile),
2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70), and
2,2'-azobis[(2-carboxyethyl)-2-(methylpropionamidine) (V-057). In
the present invention, a polymer chain is allowed to grow from this
initiator. The RAFT agent that may be used in the process is not
particularly limited and examples include benzyl dithiobenzoate,
cumyl dithiobenzoate, 2-cyanopropyl dithiobenzoate,
1-phenylethylphenyl dithioacetate, cumylphenyl dithioacetate,
benzyl 1-pyrrolecarbodithioate, cumyl 1-pyrrolecarbodithioate,
etc.
[0061] The solvent to be used during polymerization in the present
invention is not particularly limited and preferred examples are
benzene, tetrahydrofuran, 1,4-dioxane, dimethylformamide (DMF),
etc. A suitable solvent may be selected, also without any
particular limitation, as appropriate for the type of the monomer,
RAFT agent and polymerization initiator to be used in
polymerization reaction. Other factors including the concentrations
of initiator and RAFT agent during polymerization, as well as the
reaction temperature and time are not particularly limited and may
be varied depending on the object. The reaction mixture may be put
to a stationary state or stirred.
[0062] In the present invention, the thus obtained graft polymer
may be dissolved or dispersed in an organic solvent, followed by
coating this copolymer uniformly onto a substrate surface. The
solvent to be used in the process is not particularly limited as
long as it is capable of dissolving or dispersing the block
copolymer of interest and examples include N,N-dimethylacrylamide,
isopropyl alcohol, or a liquid mixture of acetonitrile and
N,N-dimethylformamide. If a plurality of solvents are to be used,
their mixing ratio is not particularly limited and the following
examples may be given: in the case of tetrahydrofuran/methanol, the
recommended ratio is one for tetrahydrofuran vs. four to six for
methanol; in the case of dioxane/normal propanol, the recommended
ratio is one for dioxane vs. four to six for normal propanol; in
the case of toluene/normal butanol, the recommended ratio is one
for toluene and four to six for normal butanol; in the case of the
acetonitrile/N,N-dimethylformamide liquid mixture, the recommended
ratio is five for acetonitrile vs. one for N,N-dimethylformamide,
or four for acetonitrile vs. one for N,N-dimethylformamide, or six
for acetonitrile vs. one for N,N-dimethylformamide.
[0063] In the present invention, the above-described graft polymer
solution need be coated uniformly onto a substrate surface. The
coating method is not particularly limited and examples include the
use of a spin coater and stationary placement of the substrate on a
horizontal table. Subsequent removal of the solvent will provide
the temperature-responsive substrate for cell culture or
temperature-responsive carrier for liquid chromatography according
to the present invention. The method of removing the solvent in the
process is not particularly limited and examples include:
evaporating the solvent slowly over time at room temperature in the
atmosphere; evaporating the solvent slowly over time at room
temperature in an environment saturated with the solvent;
evaporating the solvent under heating; and evaporating the solvent
under reduced pressure; to ensure that the finally prepared
temperature-responsive substrate for cell culture or
temperature-responsive carrier for liquid chromatography will have
a clean surface, the first two of the methods described above are
recommended and a particularly preferred method is by evaporating
the solvent slowly over time at room temperature in an environment
saturated with the solvent.
[0064] The cell culture substrate to be coated in accordance with
the present invention may be formed of any materials including not
only those which are commonly employed in cell culture such as
glass, modified glass, polystyrene and poly(methyl methacrylate)
but also those which can generally be shaped, as exemplified by
graft polymers other than those described above, ceramics, and
metals. In terms of shape, the substrate is not limited to dishes
for cell culture such as petri dish and may assume the form of a
plate, fiber or (porous) particle. If desired, it may safely assume
the shape of a vessel commonly used in cell culture, etc. (as
exemplified by a flask).
[0065] The cell to be used on the surface of the
temperature-responsive substrate for cell culture which is obtained
in the present invention may be of animal origin and the source of
its supply and the method of its preparation are not particularly
limited. Examples of the cell that may be used in the present
invention include cells derived from animals, insects, plants, etc.
as well as from bacteria. Notably, animal cells may originate from
a variety of sources including, but not particularly limited to,
humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea
pigs, pigs, sheep, Chinese hamsters, cattle, marmosets, and African
green monkeys. The culture medium to be used in the present
invention is not particularly limited as long as it is capable of
culturing animal cells and examples include a serum-free medium, a
serum-containing medium, etc. These media may be supplemented with
differentiation inducing substances such as retinoic acid and
ascorbic acid. The seeding density on the substrate surface is not
particularly limited and may be determined in the usual manner.
[0066] The temperature-responsive substrate for cell culture
according to the present invention enables cultured cells to be
exfoliated, without enzymatic treatment, by bringing the
temperature of the substrate either to a point equal to or above
the upper critical solution temperature of the
temperature-responsive polymer or to a point equal to or below its
lower critical solution temperature. This process may be performed
within a culture broth or other isotonic solutions and a suitable
way may be selected depending on the object. For the purpose of
exfoliating and recovering the cultured cells at a faster rate with
a higher efficiency, the substrate may be tapped or rocked or,
alternatively, the culture medium may be agitated with a pipette;
these and other suitable methods may be used either alone or in
combination.
[0067] By making use of the temperature-responsive substrate for
cell culture which is described herein, cells obtained from various
tissues can be cultured efficiently. Use of this culture method
enables the cultured cells to be exfoliated efficiently and intact
by simply changing the temperature. Such operations have heretofore
required time and operator's skill but according to the present
invention, this need is eliminated and mass processing of cells is
realized. In the present invention, such an improved surface of
culture substrate is prepared by utilizing living radical
polymerization and it can be designed conveniently and precisely;
by subsequently allowing the reaction to be continued on a terminal
of the molecular chain, functional groups can be conveniently
introduced and this proves extremely advantageous for cell
culture.
[0068] The carrier to be used in the present invention is not
particularly limited if it is one for use in chromatography and
polystyrene is recommended since it is capable of efficient
immobilization of the dendritic polymer having a styrene skeleton.
In this case, the pore diameter is not particularly limited and
typically ranges from 50 to 5000 .ANG. (i.e. not less than 50 .ANG.
but not more than 5000 .ANG.), preferably from 100 to 1000 .ANG.
(i.e., not less than 100 .ANG. but not more than 1000 .ANG.), and
more preferably from 120 to 500 .ANG. (i.e., not less than 120
.ANG. but not more than 500 .ANG.). Below 50 .ANG., the solutes
that can be separated are limited to those which have considerably
low molecular weights; above 5000 .ANG., the surface area of the
carrier is so small that separation efficiency will become quite
low.
[0069] In the present invention, the thusly obtained
temperature-responsive liquid chromatographic carrier is packed in
a column which is then fitted on a conventional liquid
chromatographic unit and utilized as a liquid chromatographic
system. In the process, separation in accordance with the present
invention is affected by the temperature of the carrier packed in
the column. In this case, the method of temperature loading on the
carrier is not particularly restricted and in one example, the
carrier-packed column is entirely or partially mounted on an
aluminum block, water bath, air layer, jacket or the like that have
been conditioned to a predetermined temperature.
[0070] The separation method is not particularly limited and in one
example, the carrier-packed column is held at a specified
temperature to separate the solute. The carrier to be used in the
present invention will undergo temperature-dependent variations in
the properties of its surface. Depending on the substance to be
separated, successful separation may be achieved by simply setting
the temperature at an appropriate, specified point.
[0071] In another example of the separation method, the critical
temperature at which the properties of the carrier surface will
change is preliminarily determined and solute separation is
performed by changing the temperature in such a way that it crosses
the level of that critical temperature. In this case, temperature
changes will suffice for the properties of the carrier surface to
change greatly, so depending on the solute, a great difference is
expected to occur in the time when a signal appears (i.e. retention
time). In the case of the present invention, the most effective way
of utilization is by separating the solute in such a way that the
temperature crosses the level of the critical point at which the
properties of the carrier surface will change greatly. Usually, in
the case where only the dendritic polymer is bound to the carrier
surface and the solute is a hydrophobic substance such as a
pharmaceutical product, the retention time at temperatures lower
than the critical point at which the properties of the carrier
surface will change greatly is longer than the retention time at
temperatures higher than that critical point. Presumably, for the
reason already stated above, the properties of the dendritic
polymer on the carrier surface may behave as if the surface were
hydrophobic on the lower, rather than higher, temperature side.
[0072] In the above-described process of temperature changes, the
temperature may be changed, starting from the time of starting the
solute to flow, either once or two or more times in an intermittent
or continuous manner. If desired, these methods may be combined. In
this case, temperature may be changed either manually or with the
aid of a device capable of programmed automatic temperature
control.
[0073] Alternatively, another separation method may be performed by
making use of the catch-and-release approach, in which the solute
is once adsorbed on the prepared temperature-responsive liquid
chromatographic carrier and thereafter the adsorbed solute is made
free by changing the temperature and, hence, the properties of the
carrier surface. In the process, the amount of the solute to be
adsorbed may or may not exceed the maximum amount that can be
adsorbed on the carrier. In either case, the alternative separation
method involves adsorbing the solute once and subsequently making
the adsorbed solute free by changing the temperature and, hence,
the properties of the carrier surface.
[0074] In yet another separation method, two or more
temperature-responsive liquid chromatographic carriers are packed
within the same column and the solute is separated by changing the
temperature in such a way that it crosses the level of the critical
point at which the properties of the carrier surface will change.
In this case, if the number of carriers used is two, for example,
three critical temperature zones occur where the properties of the
carrier surface will vary and temperature change may be effected by
the above-described method in such a way that the temperature will
cross the level of the critical point in each of those zones. If
desired, this may be performed with two or more
temperature-responsive liquid chromatographic carriers being packed
within two or more columns.
[0075] In still another separation method, the solute is separated
with the temperature-responsive liquid chromatographic carrier in a
column the inlet and outlet temperatures of which are so set that
the point at which the properties of the carrier surface will
change is in between said inlet and outlet temperatures and that
the temperature in the column is provided with a temperature
gradient from the inlet to the outlet end. The method of changing
the temperature stepwise is not particularly limited and examples
are by keeping the temperature in the whole column at a
predetermined level while securely monitoring the inlet and outlet
temperatures of the column or by bringing the column into contact
with a plurality of connected aluminum blocks having different
temperatures.
[0076] As described on the foregoing pages, the present invention
enables the solute to be separated by simply changing the
temperature while fixing the mobile phase. In the process, the
mobile phase is preferably a 100% aqueous system but in the case of
the present invention which depends on the properties of the
dendritic polymer immobilized on the carrier surface, the
composition of the mobile phase is not a particular limiting factor
and it may contain a solvent, its pH may be changed, or a salt may
be contained in it. In the process, the concentration of the
solvent may be changed to perform the solvent gradient method in
combination with the use of the carrier of the present invention.
If desired, the mobile phase may entirely be composed of an organic
solvent.
[0077] The temperature-responsive liquid chromatographic carrier
and the chromatographic method using the same that have been
described above according to the present invention enable
separation of pharmaceuticals and their metabolites, as well as
agrochemicals, peptides, and proteins. In the process, separation
can be accomplished by the simple operation of changing the
temperature within the column.
EXAMPLES
[0078] On the following pages, the present invention will be
described in greater detail by referring to Examples but these are
by no means intended to limit the present invention.
Example 1
[0079] (1) Synthesis of a Hyperbranched Polystyrene (HBPS)
[0080] A Schlenk flask was charged with styrene (8.05 g), CMS (5.46
g) and chlorobenzene (20 mL) and a freeze-pump-thaw cycle was
repeated three times for degasification. Subsequently,
2,2'-bipyridyl (1.64 g) and CuCl (0.520 g) were added, followed by
repeating a freeze-pump-thaw cycle three additional times for
complete degasification; thereafter, the reaction mixture was
stirred under vacuum in an oil bath (120.degree. C.). Four hours
later, the reaction was brought to an end by cooling and after
about 2-fold dilution with THF, the reaction mixture was passed
through neutral alumina to remove the copper catalyst. The reaction
mixture was further subjected to re-precipitation with hexane and
vacuum-dried overnight at 60.degree. C. to yield a pure polymer
(hereinafter referred to as HBPS). The product was analyzed by NMR
to give the result shown in FIG. 7, from which one can see that the
desired product had been obtained.
[0081] (2) Synthesis of RAFT Agent (CTA; Reversible
Addition-Fragmentation Chain-Transfer Agent)
[0082] An eggplant-shaped flask was charged with tripotassium
phosphate (1.02 g), dodecanethiol (1.34 g) and acetone (20 mL) and
after stirring the mixture for 10 minutes, carbon disulfide (1.37
g) was added and stirring was continued for an additional 10
minutes. Then, 2-bromoisobutyric acid (1.00 g) was added and,
thereafter, the mixture was stirred at room temperature for 13
hours in a nitrogen atmosphere. The solvent in the reaction mixture
was distilled off and the residue was dissolved in dichloromethane,
followed by extraction of the organic layer by phase separation
with 1M hydrochloric acid, water and saturated brine. Further
purification by silica gel chromatography gave the desired product
as a yellow crystal.
[0083] (3) Synthesis of Propargyl Terminated CTA
[0084] The above-prepared CTA (1.00 g), propargyl alcohol (0.308 g)
and DMAP (0.355 g) were put into an eggplant-shaped flask and
purged with nitrogen, followed by addition of dichloromethane (50
mL). After stirring the reaction mixture at 0.degree. C. for 30
minutes, DCC (0.567 g) dissolved in DCM (5 mL) was slowly added
dropwise and the resulting mixture was stirred for 24 hours. After
the reaction, the by-product was filtered off and the solvent was
removed; by subsequent silica gel chromatography, the desired
product of a bright, dark orange color was isolated.
[0085] (4) Block Copolymerization of PNIPAM at a Terminal of the
Hyperbranched Polystyrene
(4-1) Converting a Terminal of the Hyperbranched Polystyrene to an
Azide Form
[0086] The above-obtained HBPS and NaN.sub.3 (FIG. 3) were purged
with nitrogen and, after adding DMF (10 mL), the mixture was
stirred at 45.degree. C. for 3 days. After the reaction, DMF was
distilled off and the residue was dissolved in DCM, followed by
extraction of the organic layer by phase separation with water and
saturated brine. The extracted organic layer was vacuum-dried
overnight at 45.degree. C. Since the resulting polymer terminated
with an azide form (hereinafter referred to as HBPS-N.sub.3) was
unstable, it was dried and immediately subjected to the following
reaction.
(4-2) Modifying a Terminal of the Hyperbranched Polystyrene with
CTA (Click Reaction)
[0087] An eggplant-shaped flask was charged with the propargyl
terminated CTA obtained in step (3) and various grades of
N.sub.3-HBPS in the amounts indicated in FIG. 4 and, after nitrogen
purge together with Cu(PPh.sub.3).sub.3Br (30 mg), DMF (20 mL) was
added. After stirring at room temperature for 3 days, DMF was
distilled off. The residue was dissolved in THF and after
re-precipitation with hexane, the precipitate was vacuum-dried
overnight at 40.degree. C. to isolate the polymer modified with CTA
at a terminal (hereinafter referred to as HBPS-CTA).
(4-3) Reversible Addition-Fragmentation Chain-Transfer (RAFT)
Polymerization of PNIPAM
[0088] A 5-mL frozen ampoule was charged with AIBN (1 mg), THF (1.5
mL), NIPAM and various grades of HBPS-CTA in the amounts indicated
in FIG. 5. The system was degassed by repeating a freeze-pump-thaw
cycle four times and the mixture was stirred at 70.degree. C. for
24 hours under vacuum. After the reaction, the mixture was cooled
on an ice bath, followed by dilution with THF and re-precipitation
with hexane. The precipitate was vacuum-dried overnight at
45.degree. C. to yield the final product (HBPS-PNIPAM). Samples
50-1, 20-1 and 10-1 were analyzed by NMR to give the results shown
in FIGS. 8, 9 and 10, respectively, from which one can see that the
desired products had been obtained.
Example 2
[0089] Coating HBPS-PNIPAM onto a Cell Culture Substrate
Surface
(2-1) Preparing HBPS-PNIPAM Coating Solution
[0090] Approximately 10 mg of each sample of HBPS-PNIPAM was
collected in a sample bin and after accurately weighing the sampled
quantity, 4 ml of tetrahydrofuran/methanol (mixed at a ratio of
1:4) which would serve as the developing solvent for the coating of
HBPS-PNIPAM was added by pipetting to make a solution (hereinafter
referred to as the stock solution of polymer.) This stock solution
of polymer was divided into predetermined smaller portions which
were diluted with predetermined amounts of tetrahydrofuran/methanol
(mixed at a ratio of 1:4) to prepare diluted solutions (hereinafter
referred to as polymer coating solutions). The respective polymer
coating solutions contained HBPS-PNIPAM dissolved in amounts of
57.6 .mu.g, 48.0 .mu.g, 43.2 .mu.g, 38.4 .mu.g, 33.6 .mu.g, 28.8
.mu.g, 24.0 .mu.g, 19.2 .mu.g and 14.4 .mu.g in 50 .mu.l.
(2-2) Coating HBPS-PNIPAM onto a Surface of Cell Culture
Substrate
[0091] The respective polymer coating solutions obtained in step
(2-1) were each added dropwise in a specified amount of 50 .mu.l
over a commercial PSt petri dish (product of Becton, Dickinson
Company; Falcon 3001 with a culture area of 9.6 cm.sup.2).
Thereafter, the dishes were lidded and left to stand at room
temperature for 90 minutes, during which the solvent consisting of
tetrahydrofuran/methanol (mixed at a ratio of 1:4) evaporated
slowly to yield PSt petri dishes coated with HBPS-PNIPAM
(hereinafter referred to as temperature-responsive petri dishes).
Since the polymer coating solutions containing HBPS-PNIPAM
dissolved in amounts of 57.6 .mu.g, 48.0 .mu.g, 43.2 .mu.g, 38.4
.mu.g, 33.6 .mu.g, 28.8 .mu.g, 24.0 .mu.g, 19.2 .mu.g and 14.4
.mu.g were each applied in an amount of 50 .mu.l onto the PSt petri
dish having a culture area of 9.6 cm.sup.2, the PSt petri dishes
were coated with HBPS-PNIPAM at respective doses of 6.0
.mu.g/cm.sup.2, 5.0 .mu.g/cm.sup.2, 4.5 .mu.g/cm.sup.2, 4.0
.mu.g/cm.sup.2, 3.5 .mu.g/cm.sup.2, 3.0 .mu.g/cm.sup.2, 2.5
.mu.g/cm.sup.2, 2.0 .mu.g/cm.sup.2 and, 1.5 .mu.g/cm.sup.2.
Example 3
[0092] Evaluation of Temperature-Responsive Petri Dishes for Cell
Quality
[0093] On each of the temperature-responsive petri dishes prepared
in Example 2, 3T3 mouse fibroblasts were seeded, and each dishes
was evaluated for cell quality by examining the adhesion of cells
and by checking for the ability of cultured cells to exfoliate upon
cooling, in the early stage of culture (after one day of culture)
and after prolonged culture (after four days of culture).
(3-1) Evaluation of Temperature-Responsive Petri Dishes for Cell
Quality in the Early Stage of Culture
[0094] To each of the temperature-responsive petri dishes, 2 ml of
a medium (Dulbecco's modified Eagle's medium (DMEM) containing 10%
fetal calf serum) was added and after further adding 200 .mu.l of a
medium containing 1.times.10.sup.5 3T3 mouse fibroblasts dispersed
therein, culture was conducted in a CO.sub.2 incubator (37.degree.
C., 5% CO.sub.2) for 24 hours. After the end of culture, the
cultured cells were observed with an inverted microscope to see how
they adhered. Thereafter, the temperature-responsive petri dishes
containing the cultured cells were left to stand in a cold CO.sub.2
incubator (20.degree. C., 5% CO.sub.2) to be cooled for 15 minutes.
After the cooling, the cultured cells were observed with an
inverted microscope to see how they were exfoliated.
(3-2) Evaluation of Temperature-Responsive Petri Dishes for Cell
Quality after Prolonged Culture
[0095] To each of the temperature-responsive petri dishes, 2 ml of
a medium (Dulbecco's modified Eagle's medium (DMEM) containing 10%
fetal calf serum) was added and after further adding 200 .mu.l of a
medium containing 1.times.10.sup.5 3 T3 mouse fibroblasts dispersed
therein, culture was conducted in a CO.sub.2 incubator (37.degree.
C., 5% CO.sub.2) for 4 days. After the end of culture, the cultured
cells were observed with an inverted microscope to see how they
adhered and whether they had proliferated until confluence in the
petri dishes. Thereafter, the temperature-responsive petri dishes
containing the cultured cells were left to stand in a cold CO.sub.2
incubator (20.degree. C., 5% CO.sub.2) to be cooled for 15 minutes.
After the cooling, the cultured cells were observed with an
inverted microscope to see whether they were exfoliated in a sheet
form. Cells on the petri dish coated with 3.0 .mu.g/cm.sup.2 of
sample 50-1 appeared as shown in FIG. 11(1) at the end of culture;
the cultured cells appeared as shown in FIG. 11(2) after 15-min
cooling; cells on the petri dish coated with 3.5 .mu.g/cm.sup.2 of
sample 50-2 appeared as shown in FIG. 12(1) at the end of culture;
the cultured cells appeared as shown in FIG. 12(2) after 15-min
cooling; cells on the petri dish coated with 2.5 .mu.g/cm.sup.2 of
sample 30-1 appeared as shown in FIG. 13(1) at the end of culture;
the cultured cells appeared as shown in FIG. 13(2) after 15-min
cooling. The results show that when cultured on each sample of the
temperature-responsive substrate for cell culture according to the
present invention, cells proliferated until they became confluent
and that the cultured cells could be exfoliated in a sheet form
simply upon cooling for 15 minutes.
Comparative Example 1
[0096] Cells were cultured by the same procedure as in Example 3,
except that the cell culture substrate was a commercial product
without a coating of the temperature-responsive polymer, and an
attempt was made to exfoliate the cultured cells by simply cooling
the substrate. As it turned out, the cultured cells could not be
exfoliated from the commercial culture substrate.
Comparative Example 2
[0097] In the process of preparing sample 50-1 in step (4-3) of
Example 1, the reaction time was changed to 6 hours, yielding a
product with PNIPAM having a molecular weight of 1800. A
temperature-responsive culture substrate was prepared by coating
3.5 .mu.g/cm.sup.2 of this product in the same manner as in Example
2. On this temperature-responsive culture substrate, cells were
cultured by the same procedure as in Example 3 and an attempt was
made to exfoliate the cultured cells by simply cooling the
substrate. As it turned out, the cultured cells could not be
exfoliated from the commercial substrate.
Comparative Example 3
[0098] A temperature-responsive substrate was prepared by the same
procedure as in Example 2, except that when sample 50-1 was coated
onto a substrate surface in accordance with the method of Example
2, the coating weight was changed to 7.5 .mu.g/cm.sup.2. An attempt
was made to culture cells on the prepared temperature-responsive
culture substrate by the same procedure as in Example 3. As it
turned out, the cultured cells could not be adhered to the culture
substrate used.
Example 4
[0099] In step (4-2) of Example 1, the azide group at a terminal of
the hyperbranched polystyrene was reacted with acrylic acid to
prepare a product in which part of the terminal was converted to a
carboxyl group and by subsequently repeating step (4-3) of Example
1 and the procedure of Example 2, there was obtained a
temperature-responsive culture substrate that had a coating of
sample 50-2 in an amount of 3.5 .mu.g/cm.sup.2. On this
temperature-responsive culture substrate, cells were cultured by
the same procedure as in Example 3 and an attempt was made to
exfoliate the cultured cells by simply cooling the substrate. As it
turned out, the cells had better adhesion in the early stage of
culture and proliferated to a confluent state; the cultured cells
were exfoliated in a sheet form by simply cooling the substrate for
15 minutes.
Example 5
[0100] On a temperature-responsive culture substrate with a 3.5
.mu.g/cm.sup.2 coating of sample 50-2 that had been obtained in
accordance with the method of Example 2, a single layer of
epidermal keratinocytes was cultured in a commercial KGM medium
under a serum-free condition by the same procedure as in Example 3
and after 14 days of culture, an attempt was made to exfoliate the
cultured cells by simply cooling the substrate. As it turned out,
the cells had proliferated to a confluent state and the cultured
cells were exfoliated in a sheet form by simply cooling the
substrate for 15 minutes.
Example 6
[0101] On a temperature-responsive culture substrate with a 4.0
.mu.g/cm.sup.2 coating of sample 50-1 that had been obtained in
accordance with the method of Example 2, a single layer of retinal
pigment epithelial cells was cultured in a commercial RtEBM medium
in the presence of a serum by the same procedure as in Example 3
and after 21 days of culture, an attempt was made to exfoliate the
cultured cells by simply cooling the substrate. As it turned out,
the cells had proliferated to a confluent state and the cultured
cells were exfoliated in a sheet form by simply cooling the
substrate for 15 minutes.
Example 7
[0102] Coating HBPS-PNIPAM onto a Liquid Chromatographic Carrier
Surface
(7-1) Preparing HBPS-PNIPAM Coating Solution
[0103] Approximately 10 mg of each sample of HBPS-PNIPAM was
collected in a sample bin and after accurately weighing the sampled
quantity, 4 ml of tetrahydrofuran/methanol (mixed at a ratio of
1:4) which would serve as the developing solvent for the coating of
HBPS-PNIPAM was added by pipetting to make a solution (hereinafter
referred to as the stock solution of polymer.) This stock solution
of polymer was divided into predetermined smaller portions which
were diluted with predetermined amounts of tetrahydrofuran/methanol
(mixed at a ratio of 1:4) to prepare diluted solutions (hereinafter
referred to as polymer coating solutions.) The respective polymer
coating solutions contained HBPS-PNIPAM dissolved in amounts of
57.6 .mu.g, 48.0 .mu.g, 43.2 .mu.g, 38.4 .mu.g, 33.6 .mu.g, 28.8
.mu.g, 24.0 .mu.g, 19.2 .mu.g and 14.4 .mu.g in 50 .mu.l.
(7-2) Coating HBPS-PNIPAM onto a Liquid Chromatographic Carrier
Surface
[0104] The respective polymer coating solutions obtained in step
(7-1) were each added dropwise in a specified amount of 50 .mu.l
over commercial PSt beads for liquid chromatography that had been
preliminarily placed on a petri dish. Thereafter, the beads were
left to stand at room temperature for 90 minutes, during which the
solvent consisting of tetrahydrofuran/methanol (mixed at a ratio of
1:4) evaporated slowly to yield liquid chromatographic carriers
coated with HBPS-PNIPAM.
Example 8
[0105] Separation of Steroids
[0106] Two kinds of steroid were dissolved in purified water to
prepare 10 ml of a solution containing the steroids in admixture at
the concentrations indicated below. The prepared solution of mixed
steroids was passed through a PTFE filter (0.2 .mu.m).
Sample A
TABLE-US-00001 [0107] 1. Hydrocortisone 0.03 mg/ml 2. Testosterone
0.02 mg/ml
[0108] A column packed with the above-described packing agent
(i.e., the temperature-sensitive carrier of the present invention)
was loaded with 20 .mu.l of sample A. The column was connected to
an HPLC unit, where high-performance liquid chromatography was
conducted with a 66.7 mM phosphate buffer as a mobile phase at a
flow rate of 1 ml/min and detection was performed with UV from an
ultraviolet/visible light absorbance detector at a wavelength of
254 nm. The column was installed in a thermostatic chamber and
resolution was compared at varying column temperatures. The results
are shown in FIG. 14. At 40.degree. C., testosterone (the
right-hand peak in FIG. 14) showed a retention time of 11.5
minutes; at 30.degree. C., the retention time was 15.6 minutes; at
20.degree. C., 21.2 minutes; and at 10.degree. C., 50.9 minutes.
Thus, it was verified that changing the column temperature made it
possible to vary the hydrophobic interactions at the carrier
surface, hence, the retention time of each steroid.
INDUSTRIAL APPLICABILITY
[0109] A novel graft polymer can be obtained according to the
present invention. If this graft polymer is applied to the
temperature-responsive substrate for cell culture described herein,
cells obtained from a variety of tissues can be cultured with high
efficiency. If this culture method is utilized, cultured cells can
be exfoliated intact in a short amount of time with high
efficiency.
[0110] In addition, by using the chromatographic carrier described
herein, a wide range of peptides and proteins can also be separated
by simply changing the temperature of the chromatographic carrier.
This allows for convenient separation procedure and improves the
efficiency of separating operations. What is more, the
stereoregularity of the dendritic polymer per se may be utilized to
enable separation of solutes based on differences in their
molecular structures. The separation procedure attainable by this
method is highly likely to be applicable in the development of
pharmaceuticals, for example.
[0111] Consequently, the present invention will prove extremely
useful in a variety of fields including medicine and biology.
* * * * *