U.S. patent application number 10/552923 was filed with the patent office on 2007-04-19 for microinjection method and device.
Invention is credited to Hiroyuki Imabayashi, Sachiko Karaki, Atsushi Miyawaki.
Application Number | 20070087436 10/552923 |
Document ID | / |
Family ID | 33295854 |
Filed Date | 2007-04-19 |
United States Patent
Application |
20070087436 |
Kind Code |
A1 |
Miyawaki; Atsushi ; et
al. |
April 19, 2007 |
Microinjection method and device
Abstract
An object of the present invention is to provide a method for
introducing a physiologically active substance such as a gene into
a cell, which introduces a physiologically active substance such as
any given gene into any given cell in a view under a microscope,
while significantly reducing invasiveness to the cell, and a device
used for the above method. The present invention provides a method
for introducing a physiologically active substance into a cell,
which comprises: allowing a physiologically active substance to
attach around a needle having a diameter of 500 nm or less,
provided that it is able to be inserted into a cell; and inserting
the above-described needle into the cell; and a microinjection
device for carrying out the aforementioned method.
Inventors: |
Miyawaki; Atsushi; (Saitama,
JP) ; Imabayashi; Hiroyuki; (Tokyo, JP) ;
Karaki; Sachiko; (Tokyo, JP) |
Correspondence
Address: |
SCULLY, SCOTT, MURPHY & PRESSER, P.C.
400 GARDEN CITY PLAZA
SUITE 300
GARDEN CITY
NY
11530
US
|
Family ID: |
33295854 |
Appl. No.: |
10/552923 |
Filed: |
April 9, 2004 |
PCT Filed: |
April 9, 2004 |
PCT NO: |
PCT/JP04/05167 |
371 Date: |
August 4, 2006 |
Current U.S.
Class: |
435/455 ;
435/285.1; 977/902 |
Current CPC
Class: |
C12M 35/00 20130101;
B82Y 30/00 20130101 |
Class at
Publication: |
435/455 ;
435/285.1; 977/902 |
International
Class: |
C12N 15/00 20060101
C12N015/00; C12M 1/00 20060101 C12M001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 11, 2003 |
JP |
2003-107267 |
Claims
1. A method for introducing a physiologically active substance into
a cell, which comprises: allowing a physiologically active
substance to attach around a needle having a diameter of 500 nm or
less, provided that it is able to be inserted into a cell; and
inserting said needle into the cell.
2. The method of claim 1 wherein a needle having a diameter between
50 and 100 nm, provided that it is able to be inserted into a cell,
is used.
3. The method of claim 1 wherein a needle having a length of 5
.mu.m or less is used.
4. The method of claim 1 wherein a needle having a taper form,
provided that it is able to be inserted into a cell, is used.
5. The method of claim 1 wherein a needle composed of a carbon
nanotube is used.
6. The method of claim 1 wherein a needle composed of silicon is
used.
7. The method of claim 1 wherein a needle composed of a metal oxide
is used.
8. The method of claim 1 wherein the needle having a diameter
between 50 and 500 nm, provided that it is able to be inserted into
a cell, has electrical conductivity.
9. The method of claim 1 wherein the physiologically active
substance is DNA, RNA, or a protein.
10. The method of claim 1 wherein, using a needle charged with an
electrical charge opposite to that of a physiologically active
substance, the physiologically active substance is allowed to
electrostatically attach to said needle, and said needle is then
inserted into a cell.
11. The method of claim 1 wherein, using a needle to which a
voltage opposite to the charge of a physiologically active
substance has been applied, the physiologically active substance is
allowed to electrically attach to said needle, and said needle is
then inserted into a cell.
12. The method of claim 10 wherein after a negatively charged
physiologically active substance has been allowed to
electrostatically attach to a needle that is positively charged,
said needle is inserted into a cell, and the needle is then
negatively charged, so that the physiologically active substance is
allowed to detach from the needle.
13. The method of claim 10 wherein after a negatively charged
physiologically active substance has been allowed to.
electrostatically attach to a needle to which a positive voltage
has been applied, said needle is inserted into a cell, and a
negative voltage is then applied to the needle, so that the
physiologically active substance is allowed to detach from the
needle.
14. The method of claim 10 wherein negative voltages that change
over time are applied to the needle, so that the physiologically
active substance is allowed to detach from the needle.
15. The method of claim 14 wherein the voltages that change over
time are multiple pulse voltages.
16. The method of claim 11 wherein the needle to which a voltage
opposite to the charge of a physiologically active substance is
applied, is controlled in terms of voltage value and the time
required for application of the voltage.
17. The method of claim 1 which comprises the following steps: (1)
a step of positively charging a needle; (2) a step of immersing the
needle in a solution comprising a negatively charged
physiologically active substance, so that the physiologically
active substance is allowed to attach around the needle; (3) a step
of inserting the needle into a target site in a cell, and then
applying a negative voltage to the needle, so that the
physiologically active substance is allowed to detach from the
needle; (4) a step of removing the needle from the cell; and (5) a
step of repeating the above-described steps (1) to (4), so as to
introduce at least one desired, identical or different,
physiologically active substance into each of multiple cells.
18. A microinjection device, which comprises: a needle having a
diameter between 50 and 500 nm, provided that it is able to be
inserted into a cell; a driving means for controlling the movement
of said needle that enables insertion of said needle into the cell
and the removal therefrom; and a voltage-applying means for
applying a voltage to maintain a physiologically active substance
on the surface of said needle or to remove it from said surface,
wherein said needle is inserted into a cell and that the
physiologically active substance is then introduced into the
cell.
19. (canceled)
20. The microinjection device of claim 18 which comprises a
cell-retaining means for retaining a cell at a certain site and a
microscope for observing the cell that is retained in the
cell-retaining means.
21. The microinjection device of claim 18 which comprises a vessel
for receiving the physiologically active substance.
22. The microinjection device of claim 20 wherein the microscope
for observing the cell is provided with a means for maintaining
culture environment.
23. The microinjection device of claim 18 wherein the driving means
for controlling the movement of the needle, which is connected to
said needle, is a piezoelectric element.
24. The microinjection device of claim 18 wherein, by the driving
means for controlling the movement of the needle, said needle is
inserted into a cell from the direction of gravitational force.
25. The microinjection device of claim 18 wherein, by the driving
means for controlling the movement of the needle, said needle is
descended to a certain height with respect to the surface of the
cell-retaining means.
26. The microinjection device of claim 18 which comprises a washing
tank for eliminating the physiologically active substance attached
to the surface of the said needle.
27. The microinjection device of claim 26 wherein said washing tank
is used to perform at least one selected from sterilized water
washing, alkali washing, and acid washing.
28. The microinjection device of claim 18 wherein the time required
for application of a voltage to said needle is shorter than the
time at which said needle stays in a cell.
29. The microinjection device of claim 18 wherein said cell is
contained in a culture solution, in which physiologically active
substances are dispersed.
30. A microinjection device, which comprises: a culture solution,
in which physiologically active substances are dispersed; a
cell-retaining means for retaining a cell at a certain site; a
needle having a diameter between 50 and 500 nm, provided that it is
able to be inserted into the cell; a driving means for controlling
the movement of said needle, which is connected to the needle; and
a microscope for observing the cell retained in the cell-retaining
means; wherein said needle forms a hole that constitutes a pathway
for introducing the physiologically active substance into the
cell.
31. A method for introducing a physiologically active substance
into a cell, which comprises performing microinjection using the
microinjection device of claim 18.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for introducing a
physiologically active substance into cells and a microinjection
device used for the above method.
BACKGROUND ART
[0002] Examples of a technique of introducing gene DNA into
cultured cells or the like may include the calcium precipitation
method, the lipid transfer method, the viral vector method,
electroporation, the gene gun method, and the microinjection
method. In the above methods other than the microinjection method,
DNA is introduced in cells at a certain probability, and thus it is
impossible to introduce DNA into only a specific cell. On the other
hand, the microinjection method has been problematic in that since
the diameter of the edge of a glass pipette is approximately 1
.mu.m, cells are easily damaged when such a glass pipette is
inserted into the cell nucleus thereof. In addition, when different
genes are introduced into multiple cells, the same number of
pipettes as that of genes should be prepared, resulting in
complicated preparation.
[0003] Japanese Patent Application Laid-Open No. 2003-88383
discloses that in order to provide a means for collecting
biomolecules such as RNA from living cells, a needle capable of
specifically binding to biomolecules is inserted into a living cell
using a device enabling fine position control, and that the needle
is then removed from the cell. As a needle used herein, a ZnO
whisker or a carbon nanotube is used. For example, the surface of a
metal oxide whisker is modified with an amino group, so that the
whisker can bind to biomolecules existing in cells and collect
them.
DISCLOSURE OF THE INVENTION
[0004] As mentioned above, the conventional electroporation or gene
gun is able to inject a substance into large quantities of cells at
a time. However, it has been difficult to inject a substance into a
specific cell. Moreover, the conventional microinjection is able to
inject a substance into a specific cell. However, since a hollow
glass capillary has been used as a needle to be injected, there has
been a certain limit regarding reduction in the external diameter
thereof. Thus, these conventional methods have been problematic in
that a cell bursts or suffers fatal damage when a needle is
injected therein, or in that operations become complicated.
[0005] As described in Japanese Patent Application Laid-Open No.
2003-88383, it is possible to collect biomolecules from living
cells by performing specific modification on a metal oxide whisker
or a carbon nanotube. It is also possible to successively record a
change in each of the cells over time. However, a method for
successively recording the change in such a cell over time by
actively introducing a gene therein has not yet been disclosed. In
addition, the aforementioned method has been problematic in that
the method comprises a complicated step of modifying the surface of
a needle with a substance that is allowed to specifically bind to
biomolecules.
[0006] It is an object of the present invention to solve the
aforementioned problems of the prior art techniques. In other
words, it is an object of the present invention to provide a method
for introducing a physiologically active substance such as a gene
into a cell, which introduces a physiologically active substance
such as any given gene into any given cell in a view under a
microscope, while significantly reducing invasiveness to the cell,
and a device used for the above method.
[0007] As a result of intensive studies directed towards achieving
the aforementioned object, the present inventors have found that
the object can be achieved by using a needle having a diameter of
500 nm or less, provided that it is able to be inserted into a
cell, and by inserting the above-described needle into the cell,
thereby completing the present invention.
[0008] Thus, the present invention provides a method for
introducing a physiologically active substance into a cell, which
comprises: allowing a physiologically active substance to attach
around a needle having a diameter of 500 nm or less, provided that
it is able to be inserted into a cell; and inserting the
above-described needle into the cell.
[0009] Preferably, a needle having a diameter between 50 and 100
nm, provided that it is able to be inserted into a cell, is
used.
[0010] Preferably, a needle having a length of 5 .mu.m or less is
used.
[0011] Preferably, a needle having a taper form, provided that it
is able to be inserted into a cell, is used.
[0012] Preferably, a needle composed of a carbon nanotube is
used.
[0013] Preferably, a needle composed of silicon is used.
[0014] Preferably, a needle composed of a metal oxide is used.
[0015] Preferably, a needle having a diameter between 50 and 500
nm, provided that it is able to be inserted into a cell, has
electrical conductivity.
[0016] Preferably, the physiologically active substance is DNA,
RNA, or a protein.
[0017] Preferably, using a needle charged with an electrical charge
opposite to that of a physiologically active substance, the
physiologically active substance is allowed to electrostatically
attach to the above-described needle, and the above-described
needle is then inserted into a cell.
[0018] Preferably, using a needle to which a voltage opposite to
the charge of a physiologically active substance has been applied,
the physiologically active substance is allowed to electrically
attach to the above-described needle, and the above-described
needle is then inserted into a cell.
[0019] Preferably, after a negatively charged physiologically
active substance has been allowed to electrostatically attach to a
needle that is positively charged, the above-described needle is
inserted into a cell, and the needle is then negatively charged, so
that the physiologically active substance is allowed to detach from
the needle.
[0020] Preferably, after a negatively charged physiologically
active substance has been allowed to electrostatically attach to a
needle to which a positive voltage has been applied, the
above-described needle is inserted into a cell, and a negative
voltage is then applied to the needle, so that the physiologically
active substance is allowed to detach from the needle.
[0021] Preferably, negative voltages that change over time are
applied to the needle, so that the physiologically active substance
is allowed to detach from the needle.
[0022] Preferably, the voltages that change over time are multiple
pulse voltages.
[0023] Preferably, the needle to which a voltage opposite to the
charge of a physiologically active substance is applied, is
controlled in terms of voltage value and the time required for
application of the voltage.
[0024] Preferably, the method of the present invention comprises
the following steps:
(1) a step of positively charging a needle;
(2) a step of immersing the needle in a solution comprising a
negatively charged physiologically active substance, so that the
physiologically active substance is allowed to attach around the
needle;
(3) a step of inserting the needle into a target site in a cell,
and then applying a negative voltage to the needle, so that the
physiologically active substance is allowed to detach from the
needle;
(4) a step of removing the needle from the cell; and
(5) a step of repeating the above-described steps (1) to (4), so as
to introduce at least one desired, identical or different,
physiologically active substance into each of multiple cells.
[0025] In another aspect, the present invention provides a
microinjection device, which comprises: a needle having a diameter
between 50 and 500 nm, provided that it is able to be inserted into
a cell; a driving means for controlling the movement of the
above-described needle that enables insertion of the
above-described needle into the cell and the removal therefrom; and
a voltage-applying means for applying a voltage to maintain a
physiologically active substance on the surface of the
above-described needle or to remove it from the above surface,
wherein the above-described needle is inserted into a cell and that
the physiologically active substance is then introduced into the
cell.
[0026] In another aspect, the present invention provides a
microinjection device used for the aforementioned method of the
present invention, which comprises: a needle having a diameter
between 50 and 500 nm, provided that it is able to be inserted into
a cell; a driving means for controlling the movement of the
above-described needle that enables insertion of the
above-described needle into the cell and the removal therefrom; and
a voltage-applying means for applying a voltage to maintain a
physiologically active substance on the surface of the
above-described needle or to remove it from the above surface,
wherein the above-described needle is inserted into a cell and that
the physiologically active substance is then introduced into the
cell.
[0027] Preferably, a microinjection device, which comprises a
cell-retaining means for retaining a cell at a certain site and a
microscope for observing the cell that is retained in the
cell-retaining means, is provided.
[0028] Preferably, a microinjection device, which comprises a
vessel for receiving the physiologically active substance, is
provided.
[0029] Preferably, the microscope for observing the cell is
provided with a means for maintaining culture environment.
[0030] Preferably, the driving means for controlling the movement
of the above-described needle, which is connected to the needle, is
a piezoelectric element.
[0031] Preferably, by the driving means for controlling the
movement of the above-described needle, the needle is inserted into
a cell from the direction of gravitational force.
[0032] Preferably, by the driving means for controlling the
movement of the above-described needle, the needle is descended to
a certain height with respect to the surface of the cell-retaining
means.
[0033] Preferably, a microinjection device which comprises a
washing tank for eliminating the physiologically active substance
attached to the surface of the above-described needle, is
provided.
[0034] Preferably, the above-described washing tank is used to
perform at least one selected from sterilized water washing, alkali
washing, and acid washing.
[0035] Preferably, the time required for application of a voltage
to the above-described needle is shorter than the time at which the
above-described needle stays in a cell.
[0036] Preferably, the above-described cell is contained in a
culture solution, in which physiologically active substances are
dispersed.
[0037] In another aspect, the present invention provides a
microinjection device, which comprises: a culture solution, in
which physiologically active substances are dispersed; a
cell-retaining means for retaining a cell at a certain site; a
needle having a diameter between 50 and 500 nm, provided that it is
able to be inserted into the cell; a driving means for controlling
the movement of the above-described needle, which is connected to
the needle; and a microscope for observing the cell retained in the
cell-retaining means; wherein the above-described needle forms a
hole that constitutes a pathway for introducing the physiologically
active substance into the cell.
[0038] In another aspect, the present invention provides a method
for introducing a physiologically active substance into a cell,
which comprises performing microinjection using the aforementioned
microinjection device of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 shows a summary of the method of the present
invention.
[0040] FIG. 2 shows a microinjection device constructed on the
stage of an inverted microscope.
[0041] FIG. 3 is a top view of the microscope stage (the internal
view of an incubator).
[0042] FIG. 4 shows the positions of the microinjection device and
of the needle used for gene introduction.
[0043] FIG. 5 shows the alternate voltage .+-.5 V at 100 Hz that is
used as a voltage to be applied.
[0044] FIG. 6 shows a voltage waveform obtained during the period
ranging from the retention of gene DNA to the release thereof.
[0045] FIG. 7 shows the state of cells that are adjacent to each
other.
[0046] FIG. 8 shows an example of the voltage pattern of the
applied voltage.
[0047] FIG. 9 shows another example of the voltage pattern of the
applied voltage.
[0048] FIG. 10 shows a schematic view showing a case where gene DNA
to be introduced into a cell is dispersed in a culture
solution.
[0049] FIG. 11 shows a needle composed of a carbon nanotube, which
has a diameter of 50 nm and a length of 3 .mu.m.
[0050] FIG. 12 shows a needle produced by narrowing the diameter of
a cantilever made from silicon by etching, and forming a platinum
layer on the surface thereof.
[0051] In the above drawings, 1 represents a cantilever, 2
represents a needle, 3 represents a cell, 4 represents a cell
nucleus, 5 represents a petri dish, 6 represents a cell-retaining
means, 7 represents a vessel, 8 represents a solution containing a
physiologically active substance, 9 represents a driving means, 10
represents an electric potential-controlling means, 11 represents a
microinjection device, 12 represents an incubator, 13 represents a
heater, 14 represents a fan, 15 represents an object glass, 16
represents a specimen, 17 represents a light source used for
transillumination, 18 represents a vessel, 19 represents a washing
tank, 20 represents an XY stage, 21 represents a needle, 22
represents a stacked piezoelectric actuator, 23 represents a fixed
block, 24 represents a Z-axis stage, 25 represents the bottom of a
petri dish, 26 represents a cell, 27 represents a cell nucleus, 28
represents gene DNA, and 29 represents a pinhole.
BEST MODE FOR CARRYING OUT THE INVENTION
[0052] The embodiments of the present invention will be described
in detail below.
[0053] In the method of the present invention, a physiologically
active substance is allowed to attach around a needle having a
diameter of 500 nm or less, and the above needle is then inserted
into a cell, so as to introduce the physiologically active
substance into the cell.
[0054] The present invention is characterized in that an extremely
thin needle (to such an extent that it exceeds optical resolution)
is used for gene introduction. Specifically, a needle having a
diameter of 500 nm or less, and particularly preferably having a
diameter between 50 and 100 nm, can be used, provided that it is
able to be inserted into a cell. The needle used in the present
invention is preferably a needle, the electrical properties of
which, such as electrification, can easily be controlled. In the
present invention, for example, the surface of the needle is
positively charged, and DNA molecules are allowed to attach to the
surface. Thereafter, the needle is inserted into the cell nucleus,
and the surface of the needle is then negatively charged, so as to
allow the DNA molecules to detach from the surface of the needle.
Since a thin needle is used in the present invention, damage given
to the cell can be reduced to a minimum, and further, any given DNA
can be introduced into any given target cell.
[0055] It has been known that when cell organelle (Golgi body,
mitochondrion, and the like) is damaged with a needle, the survival
rate of the cell decreases. When a needle having a diameter between
50 and 500 nm is used, provided that it is able to be inserted into
a cell, such a needle is small enough with respect to the size of a
cell nucleus, and thus it hardly hurts cell organelle other than
the cell nucleus. Moreover, by allowing the needle to proceed to
the cell from the position directly above (from the direction of
gravitational force), the needle can be inserted into a site at
which the cell nucleus is closest to the cell membrane. Herein, the
probability that the cell organelle exists in a very small space
between the cell nucleus and the cell membrane is low. From this
viewpoint as well, the cell organelle is hardly damaged, and thus
the survival rate of cell can be improved.
[0056] In the present invention, only the use of a needle having a
surface with conductivity (electrification) is required.
Modification of the surface of the needle depending on biomolecules
is not particularly necessary.
[0057] For example, 100 types of DNA solutions and 100 cells are
prepared. Thereafter, a needle is immersed in such a DNA solution,
and the needle is then inserted into such a cell from the position
directly above the cell. Such an operation to immerse a needle in a
DNA solution and an operation to insert the needle into the cell
are repeated, so that desired individual DNAs can be introduced
into different cells, and so that the cells can individually be
transformed. Therefore, according to the method of the present
invention, screening of an agent or exhaustive analysis of
interaction between biomolecules can be carried out at a single
cell level, differing from the conventional methods wherein such
screening or analysis is carried out at a well level, using a
96-well plate or 384-well plate.
[0058] The material of the needle used in the present invention is
not particularly limited, as long as it has the aforementioned
properties. For example, a carbon nanotube can be used as a needle.
Such a carbon nanotube has a cylindrical form obtained by winding a
monolayer graphite (graphin), and it is a microcrystal composed of
100% carbon atoms. In recent years, nanotechnology has become a
focus of attention, and such a carbon nanotube has also received
attention in various fields. Examples of studies regarding the use
of a carbon nanotube include the development of a screen in which a
nanotube is used for electron gun, in place of liquid crystal or
plasma display; application of a carbon nanotube to fuel cells and
solar cells; and the use as a material for hydrogen storage. A
carbon nanotube can be applied to the aforementioned techniques
because it has various types of characteristic properties such as
minuteness, the properties as a quantum obtained from its
three-dimensional structure, and its composition purely consisting
of carbons, and thus because it has unique properties different
from those of the conventional materials. In addition, a carbon
nanotube purely consists of carbons. Thus, differing from carbon
black and the like, it contains almost no impurities. Moreover, a
carbon nanotube is also characterized in that it does not change
even after it has been exposed to a high temperature during a
molding process and/or when it is used.
[0059] At present, as a multi wall carbon nanotube, a carbon
nanotube having a diameter between approximately 50 and 100 nm and
a length of 3 .mu.m or more is available. It is preferable to use
such a carbon nanotube in the present invention. If the diameter of
a needle is too thin, the amount of a physiologically active
substance that can be retained by the needle decreases. In
contrast, if the diameter is too thick, the invasiveness to a cell
increases. Thus, both cases are unfavorable. Accordingly, in the
present invention, a needle having a diameter of 500 nm or less,
and more preferably a diameter between 50 and 100 nm, is used,
provided that it is able to be inserted into a cell. With regard to
the length of a needle, since the height of a common cultured cell
is approximately 5 .mu.m, a needle having a length of 5 .mu.m or
less can appropriately be used. For example, a needle having a
length of approximately 3 .mu.m can be used.
[0060] Except for the aforementioned examples, the following
needles can also be used.
[0061] A needle, which is produced by coating a metal oxide whisker
shown in the aforementioned example of prior art technique with
gold (Au) or platinum (Pt) using evaporation or sputtering device
or the like, so as to impart electric conductivity to the surface
thereof, can be used. Moreover, a needle, which is produced by
coating a cantilever made from silicon that has frequently been
used as a cantilever for an atomic force microscope with gold (Au)
or platinum (Pt) using evaporation or sputtering device or the
like, so as to impart electric conductivity to the surface thereof,
can also be used. The needlepoint of such a cantilever made from
silicon is etched using a device such as IPC or FIB for
acumination, and a conductive membrane is then formed thereon,
thereby further reducing the invasiveness to living cells.
Furthermore, in the case of a cantilever made from silicon, the
needlepoint thereof is converted to a taper form by etching,
thereby improving the strength of the needle. In order to reduce
damage given to a cell, it is necessary for the aforementioned
needle to have a diameter between 50 and 500 nm, provided that it
is able to be inserted into the cell. In the case of the
aforementioned needle having a taper form also, the needle has a
diameter between 50 and 500 nm, provided that it is able to be
inserted into the cell.
[0062] The type of a physiologically active substance that can be
introduced into a cell by the method of the present invention is
not particularly limited. Examples thereof may include nucleic
acids such as DNA or RNA and proteins. A preferred example may be a
nucleic acid. As such a nucleic acid, either DNA or RNA may be
used. In addition, examples of DNA used herein may include genomic
DNA or a fragment thereof, cDNA, and synthetic DNA such as a
synthetic oligonucleotide.
[0063] In the present invention, a needle having a diameter between
50 and 500 nm provided that it is able to be inserted into a cell
as mentioned above (that is, a carbon nanoprobe or a metal oxide
whisker having a conductive surface), is attached to the tip of the
cantilever of an atomic force microscope (AFM), thereby producing
electrical connection. The term "electrical connection" is herein
used to mean electrical connection for positively or negatively
controlling the charge of the needle. This. cantilever works with
the image processing of the microscope and moves between a vessel
containing a physiologically active substance of interest and a
cell of interest (a cell nucleus or the like), so that the
physiologically active substance of interest can be introduced into
only the cell of interest. In a preferred embodiment of the present
invention, the needle is always positioned in the vertical
direction, and it controls its needlepoint position at high
accuracy.
[0064] The aforementioned movement of the needle can be conducted
by a driving means for controlling the movement of the needle. That
is to say, the present invention provides a microinjection device
comprising: a needle having a diameter between 50 and 500 nm,
provided that it is able to be inserted into a cell; and a driving
means for controlling the movement of the above needle. More
specifically, the microinjection device of the present invention
comprises: (a) a cell-retaining means for retaining a cell at a
certain site; (b) a needle having a diameter between 50 and 500 nm,
provided that it is able to be inserted into the cell, and a
driving means for controlling the movement of the above needle,
which is connected to the above needle; and (c) a microscope for
observing the cell retained in the cell-retaining means.
[0065] In the present invention, using a needle charged with an
electrical charge opposite to that of a physiologically active
substance, the physiologically active substance may be allowed to
electrostatically (electrically) attach to the above needle, and
the needle may be then inserted into a cell. When a physiologically
active substance having a negative electric charge, such as DNA, is
introduced into a cell, the above physiologically active substance
is allowed to electrostatically (electrically) attach to a needle
that is positively charged, and the needle may be then inserted
into the cell.
[0066] In the present invention, utilizing the electrical polarity
of a physiologically active substance, such a physiologically
active substance may be allowed to attach to the surface of a
needle for a certain period of time, or may be allowed to detach
therefrom. That is, needless to say, such a physiologically active
substance may be not only allowed to electrostatically attach to
the surface of a needle, but it may be also allowed to attach
thereon even in a state where a voltage is continuously applied to
the needle. Thus, it is also possible that the physiologically
active substance may be allowed to detach from the surface of the
needle by reversing (inversing) the polarity of the voltage
applied.
[0067] As mentioned above, a method for applying a voltage to a
needle is not necessarily limited to electrostatic action, but it
can be selected depending on the electrical properties of the
physiologically active substance or the purpose of use. Thus, the
present invention can be applied to various purposes.
[0068] As an example, the method of the present invention comprises
the following steps:
(1) a step of positively charging a needle;
(2) a step of immersing the needle in a solution containing a
negatively charged physiologically active substance, so that the
physiologically active substance is allowed to attach around the
needle;
(3) a step of inserting the needle into a target site in a cell, so
as to introduce the physiologically active substance into the
cell;
(4) a step of removing the needle from the cell, and then
negatively charging the needle, so as to eliminate the
physiologically active substance remaining around the needle;
and
(5) a step of repeating the above-described steps (1) to (4), so as
to introduce at least one desired, identical or different,
physiologically active substance into each of multiple cells.
[0069] Hereafter, an example of the embodiments of the present
invention will be described with reference to drawings.
[0070] FIG. 1 shows a summary of the method of the present
invention. FIG. 1 shows with a double-headed arrow that a needle 2
equipped in a cantilever 1 that is connected to a driving means 9
moves between the position directly above a cell 3 and the position
directly above a vessel 7 comprising a solution 8 containing a
physiologically active substance. The cell 3 is cultured in a petri
dish 5, and the petri dish 5 is placed on a cell-retaining means
6.
[0071] First, the needle 2 is inserted into the vessel 7 comprising
the solution 8 containing a physiologically active substance, so
that the solution containing the physiologically active substance
is allowed to attach to the surface of the needle 2. Such
attachment of the above solution takes place as a result of the
control of the charge of the needle by an electric
potential-controlling means, which is electrically connected to the
needle 2. Namely, when the physiologically active substance is a
negatively charged substance such as a nucleic acid, the
physiologically active substance is allowed to efficiently attach
to the needle 2 by positively charging the needle 2 by the electric
potential-controlling means 10 in advance.
[0072] Subsequently, the needle 2, to which the physiologically
active substance has been attached, is lifted up, so that it is
removed from the vessel 7 comprising the solution 8 containing the
physiologically active substance. Thereafter, the needle moves in
the horizontal direction, and it is then disposed at the position
directly above the cell of interest 3. The needle 2 disposed at the
position directly above the cell 3 moves downwards, so that it can
be inserted into a cell nucleus 4 in the cell of interest 3. The
needle inserted into the cell nucleus 4 then releases the
physiologically active substance attached to the surface thereof to
the inside of the cell nucleus 4 under the status quo. The
physiologically active substance can be released by controlling the
charge of the needle by the electric potential-controlling means
10, which is electrically connected to the needle 2. Namely, when
the physiologically active substance is a negatively charged
substance such as a nucleic acid, the needle 2 is negatively
charged by the electric potential-controlling means 10, so that the
physiologically active substance can efficiently be released from
the needle 2. After the physiologically active substance has been
released to the inside of the cell nucleus 4, the needle is removed
from the cell. Thereafter, the aforementioned operations are
repeated, so as to introduce the desired physiologically active
substance into the desired cell nucleus. The aforementioned
movements of the needle 2 are all controlled by the driving means
9.
[0073] The present invention further relates to a microinjection
device, which comprises: a needle having a diameter between 50 and
500 nm, provided that it is able to be inserted into a cell; a
driving means for controlling the movement of the above needle that
enables insertion the above needle into the cell and the removal
therefrom; and a voltage-applying means for applying a voltage so
as to retain a physiologically active substance on the surface of
the above needle or to remove it from the surface thereof, wherein
the above needle is inserted into the cell, so as to introduce the
physiologically active substance into the cell.
[0074] The above microinjection device will be described in detail
below.
[0075] As shown in FIG. 2, the microinjection device is constructed
on the stage of an inverted microscope, enabling the observation
and/or measurement of the process from initiation of gene
introduction to the subsequent course. On the microscope stage, an
incubator 12 for maintaining the temperature at 37.degree. C. is
constructed, and the microinjection device is installed in the
incubator.
[0076] FIG. 3 is a top view of the microscope stage (the internal
view of an incubator). The incubator is composed of a metal having
excellent heat conductivity (an aluminum alloy or the like). A
heater 13 or a fan 14 for stirring the internal air is disposed on
the internal side of the incubator. The external surface of the
incubator is covered with a heat insulator in order not to release
the heat to the external environment. In addition, in order to
observe the inside of the incubator with an inverted microscope,
some regions on the top and bottom surfaces of the incubator are
made from glass, so that a specimen 16 (a cell in a petri dish or
the like) can be observed with an object glass 15. Moreover, a
light from a light source for transillumination can be applied to
the specimen, so that phase difference observation or differential
interference observation can be conducted.
[0077] In order to optimize the pH of a cell culture solution to
the culture environment, 5% CO.sub.2 is supplied from the outside
of the incubator through a tube, and using a fan, the inside of the
incubator is uniformly filled with 5% CO.sub.2.
[0078] On the microscope stage formed in the incubator, a petri
dish acting as a specimen (a dish, a microplate, or the like), a
vessel 18 comprising a solution containing gene DNA, such as a
sample cup, a washing tank 19 for washing the needle, and the like,
are placed on an XY stage 20 that is operated with a motor or the
like.
[0079] Accordingly, the specimen 16, the vessel 18, and the washing
tank 19 are able to move under a needle used for gene introduction.
Thus, as stated above, the whole area in the specimen can be
observed.
[0080] A needle 21 used for gene introduction moves only in the Z
direction (in the direction of gravitational force), and thus, it
moves up and down with respect to the target that is positioned
under the needle. As shown in FIG. 4, the needle 21 used for gene
introduction is disposed, with the needlepoint thereof directed
downwards, with respect to one end face of a stacked piezoelectric
actuator 22 formed by lamination of thin piezoelectric elements
(lead zirconate titanate). The other end face of the stacked
piezoelectric actuator 22 is disposed on a fixed block 23. When a
voltage is applied to the stacked piezoelectric actuator 22, the
needle 21 slightly moves down. A voltage of approximately 100 V
realizes a movement of 10 .mu.m, although it depends on the type of
a commercially available stacked piezoelectric actuator. Such
amount of displacement can be controlled with the value of the
voltage applied.
[0081] Further, the fixed block 23 is mounted on a Z-axis stage 24.
With regard to the position of the needle 21 in the Z direction, by
a two-step driving mechanism in which rough movements are
controlled with the Z-axis stage 24 and fine movements are
controlled with the stacked piezoelectric actuator 22, the needle
21 is inserted into the cell 26. For example, the needle 21 is
positioned above a petri dish acting as the specimen 16, it roughly
moves to the vicinity of the cell 26 in the petri dish, and
thereafter, when the needle 21 is inserted into the cell 26, it
finely moves. Since the needle 21 is very easily broken, the
movement is terminated immediately before the needlepoint is
allowed to come into contact with the bottom of the specimen 16
such as a petri dish (for example, at a height of 1 .mu.m from the
bottom).
[0082] That is to say, the needle 21 may pass through a cell
nucleus 27. This microinjection device can easily be automated by
allowing the device to recognize only the step of constantly
lifting down the needlepoint to a height of 1 .mu.m from the
bottom. In particular, the control action to detect the surface of
a cell membrane and lift down the needlepoint several .mu.m from
the position of the above surface is unnecessary, and thus
expensive detection components can be reduced.
[0083] On the other hand, when the needle is lifted down to the
vessel 18 filled with a gene DNA solution or to the washing tank
19, strict height control is unnecessary, and thus only the rough
movement is applied by the Z-axis stage 24.
[0084] The movement of the microinjection device is as described
above. It comprises the following steps.
(1) Washing of the Needle
[0085] The washing tank 19 is positioned under the needle 21 for
gene introduction, and thus the needle 21 is lifted down into the
washing tank 19. Washing solution (sterilized water) or the like is
stored in the washing tank 19. An alternate voltage is applied to
the needle 21 in a state where the needle is completely immersed in
the washing solution. Thus, dusts or gene DNA that has previously
been allowed to attach to the needlepoint are eliminated thereby.
For example, as shown in FIG. 5, the alternate voltage .+-.5 V at
100 Hz is applied as a voltage to be applied. Thereby, impurities
remaining on the surface of the needle are eliminated. Preferably,
the washing tank 19 may be subjected to ultrasonic washing, or two
tanks may be established to wash with agents such as acid or alkali
and also to wash with sterilized water.
(2) Thereafter, the needle 21 is lifted up from the washing tank
19. During such a step, the needlepoint may be dried by air blowing
or the like.
[0086] (3) Subsequently, the vessel 18 containing a gene DNA
solution moves under the needle 21, and the needle is then lifted
down therein. A positive voltage is applied to the surface of the
needle in a state where the needle is immersed in the solution. For
example, a voltage to be applied is set at 1 V, and the time
required for application of such a voltage is set to be 3 seconds
or more. Since the gene DNA has a negative polarity, it is allowed
to attach to the surface of the needle. Thereafter, the needlepoint
is lifted up, and the specimen 16 is disposed under the needle.
During this step, a voltage may be applied to or may not be applied
to the needlepoint.
[0087] (4) When the needlepoint is lifted down into a petri dish of
the specimen 16, application of the voltage to the needle 21 is
terminated. The needlepoint is lifted down to the vicinity of the
cell surface by rough movement, and thereafter, the needle 21 is
inserted into the cell nucleus 27 by fine movement.
[0088] (5) After the movement of the needle 21 has been terminated,
a negative voltage is applied to the needlepoint, so that the gene
DNA on the surface of the needle is allowed to detach therefrom,
thereby being released into the cell (into the cell nucleus). For
example, a voltage to be applied is set to be -0.5 V, and the time
required for application of such a voltage is set to be
approximately 1 second. (It is desired to apply a voltage for a
time sufficient for the gene DNA attached to the needle to be
detached therefrom.) FIG. 6 shows a voltage waveform obtained
during the period ranging from the retention of the gene DNA to the
release thereof.
[0089] (6) After completion of the application of the voltage, the
needle 21 is lifted upwards, and the needle 21 is then washed in
the washing tank 19. Thereafter, different gene DNA is allowed to
attach to the surface of the needle in a vessel containing the
different gene DNA, and it is then released into another cell.
[0090] Thus, when gene DNA attached to the surface of the needle 21
is released into a cell, a negative voltage is applied to the
needle, only when the needle 21 stays in the cell, so that the gene
DNA can be detached from the needle by electric repulsion. When a
voltage is applied to the needle in a culture solution, there are
concerns about formation of bubbles as a result of an
electrochemical reaction. Thus, in a culture solution, it is
desired to apply a voltage only for a necessary time. Accordingly,
it is adequate that a voltage be applied only when the needle stays
in the cell, and that such application of the voltage be terminated
during a travel time necessary for the needle to be inserted into
the cell and another travel time necessary for the needle to be
removed from the cell.
[0091] By repeating these operations, it becomes possible to
introduce each different gene DNA into cells 26 adjacent to each
other, as shown in FIG. 7, and this technique can be used for the
analysis of interaction between cells and the like. With regard to
the conventional microinjection device using a glass tube, the
users have manually carried out the aspiration and elimination of a
gene DNA solution by a hydraulic mechanism or the positioning of
the needlepoint to the cell, and thus a certain level of skill has
been required. However, with the structure of the present
invention, the cell nucleus 27 is recognized using the image
processing of the cell, and the needlepoint is positioned in the
center of the cell nucleus 27. The subsequent operations are easily
automated. Thus, the present invention requires no such level of
skill.
[0092] In the aforementioned embodiment, a step of introducing
different gene DNA into each cell is described. However, since the
needle 21 is extremely thin, causing no substantial damage to the
cell, multiple gene DNAs can also be introduced into a single
cell.
[0093] In addition, the voltage to be applied to the needlepoint is
not limited to the aforementioned example. The voltage patterns
shown in FIGS. 8 and 9 may also be applied, for example. In FIG. 8,
the time required for retaining gene DNA is reduced, and the amount
of such gene DNA attached to the needlepoint can thereby be
reduced. That is, the amount of gene DNA to be introduced depends
on the amount of gene DNA attached to the needlepoint. Thus, if the
voltage value is decreased and the time required for application of
the voltage is reduced, the amount of gene DNA attached is reduced.
On the contrary, if the opposite operation is carried out, the
amount attached can be increased, and the amount introduced can
thereby also be increased. These operations are effective as means
for changing the level of gene introduction to cells (making
difference in the level of gene introduction to cells). In
addition, in FIG. 9, when gene DNA is released into the cell
nucleus, pulse voltages are applied to the needlepoints at short
intervals (voltages that change over time), so that the action of
removing gene DNA attached to the needle therefrom can be promoted,
and so that almost all amount of gene DNA can be removed. It cannot
be said that application of voltages has no influence upon the
cell, and it may become stimulation. Accordingly, the shorter the
time required for application of voltages, more preferable it is.
For example, a voltage to be applied is set to be -1 V, and 10
pulses of such voltages are applied at 10 Hz.
[0094] The stacked piezoelectric actuator 22 used for the fine
movement of the needle can be displaced at a high speed,
substantially depending on application of a voltage. For example,
the natural frequency of a stacked piezoelectric actuator 22 having
a cross section of 5 mm square and a length of 20 mm in the bending
direction is several kHz. The needlepoint moves in response to the
voltage pattern at less than the above frequency (less than the
resonant region). Thus, the needle 21 is inserted into the cell 26
at a high speed of several Hz, and the needle 21 is then lifted
upwards at that cycle. By applying the aforementioned multiple
pulse voltages during the short time from insertion to removal, the
time required for the needle 21 to stay in the cell 26 can also be
reduced, thereby reducing the invasiveness of the needle to the
cell 26.
[0095] A case where gene DNA to be introduced into a cell is
dispersed in a culture solution, as shown in FIG. 10, will be
described below. The needle 21 used for gene introduction is moved
up and down at a higher speed (1 cycle frequency consisting of
several kHz) by the stacked piezoelectric actuator 22, so as to
form a pinhole 29 on the cell membrane (cell nucleus 27). As
described above, since the needle 21 has a very small diameter,
insertion of such a needle has only little influence upon the cell
26. Moreover, by inserting and removing the needle 21 at a high
speed, damage to the cell 26 can be further reduced. Thereby, a
pathway for introducing gene DNA contained in a solution into the
cell 26 (into the cell nucleus 27) is secured. By performing
culture in such a state, the gene DNA in the solution can be
introduced into the cell. Accordingly, by forming one or more
pinholes in each of the cells 26 by automatic movement, gene DNA
can easily and simply be introduced into multiple cells.
[0096] Preferably, a positive voltage is applied to the needle in a
culture solution, in which gene DNA 28 is dispersed, and the gene
DNA is retained on the surface of the needle. Multiple negative
pulse voltages are applied during the time at which the needle is
inserted into the cell, so as to release the gene DNA into the
cell. The needle moves in the XY face in the culture solution, so
that the gene DNA can successively be introduced into different
cells. The operation to immerse the needle in a vessel so as to
retain gene DNA on the surface of the needle, the washing
operation, and the like, can be omitted, and thereby such gene DNA
can be introduced into a large number of cells. Thus, the
introduction efficiency is also increased.
[0097] Thereafter, the cell, into which a gene has been introduced
as mentioned above, is continuously cultured in an incubator
equipped in a microscope, thereby enabling observing and/or
measuring over time, the process up to the expression of the gene,
or the interaction between cells.
[0098] The present invention will be more specifically described in
the following examples. However, the examples are not intended to
limit the scope of the present invention.
EXAMPLES
Example 1
[0099] Using the device shown in FIG. 1, DNA was introduced into
nerve cells that were cultured in a petri dish for culture.
[0100] As nerve cells, PC12. cells (nervous system clone cells
isolated from rat adrenal medulla pheochromocytoma) were used. As a
medium, DMEM (Dulbecco's Modified Eagle Medium) containing 10%
fetal bovine serum (FBS) was used. The culturing was carried out at
37.degree. C. in 5% CO.sub.2. As DNA, a recombinant expression
vector containing NGF receptor gene was used, and 1 .mu.g/ml DNA
solution was used.
[0101] The needle used for the device shown in FIG. 1 composed of
the carbon nanotube shown in FIG. 11, which had a diameter of 50 nm
and a length of 3 .mu.m.
[0102] First, the needle was immersed in the DNA solution, so as to
allow DNA to attach to the surface thereof. Thereafter, the needle
was inserted into the nucleus of a nerve cell, and then released
the DNA therein. After completion of the introduction of the DNA,
the nerve cell was continuously cultured. Even 3 days later, the
nerve cell still survived.
[0103] In the case of the needle shown in FIG. 12, the cantilever
thereof made from silicon was etched for acumination, and a
platinum layer was formed on the surface thereof. Using this
needle, DNA was introduced into Hela cells. As a result, it was
found that even 3 days after DNA introduction, the Hela cells still
survived.
[0104] On the other hand, as a control, microinjection was carried
out using a glass pipette (inside diameter: 300 .mu.m) filled with
the same above DNA solution, instead of using the needle composed
of a carbon nanotube with a diameter of 50 nm and a length of 3
.mu.m. After completion of the microinjection, the nerve cells were
continuously cultured. However, the nerve cells died until 3 days
later, and no surviving cells existed.
INDUSTRIAL APPLICABILITY
[0105] The present invention provides a method for introducing a
physiologically active substance such as any given gene into any
given cell that is in a view under a microscope, while
significantly reducing invasiveness to the cell, and a device used
for the above method.
* * * * *