U.S. patent number RE38,323 [Application Number 09/169,188] was granted by the patent office on 2003-11-18 for cell potential measurement apparatus having a plurality of microelectrodes.
This patent grant is currently assigned to Matsushita Electric Industrial Co., Ltd.. Invention is credited to Akihito Kamei, Yasushi Kobayashi, Tadayasu Mitsumata, Hirokazu Sugihara, Makoto Taketani.
United States Patent |
RE38,323 |
Sugihara , et al. |
November 18, 2003 |
Cell potential measurement apparatus having a plurality of
microelectrodes
Abstract
A cell potential measurement apparatus, which uses a planar
electrode enabling a multi-point simultaneous measurement of
potential change arising from cell activities, is provided which
can conduct measurements accurately and efficiently as well as can
improve convenience of arranging measurement results. According to
the configuration of the cell potential measurement apparatus of
this invention, it includes an integrated cell holding instrument,
which includes a planar electrode provided with a plurality of
microelectrodes arranged in a matrix form on the surface of a
substrate, a cell holding part for placing cells thereon, drawer
patterns from the microelectrodes, and electric contact points for
outside connections; an optical observation means for optical
observations of cells; a stimulation signal supply means to be
connected to the cell holding instrument for providing electric
stimulation to the cells; and a signal processing means to be
connected to the cell holding instrument for processing an output
signal arising from electric physiological activities of the cells.
It is preferable that a cell culturing means is also provided for
maintaining a culture atmosphere of the cells placed on the
integrated cell holding instrument.
Inventors: |
Sugihara; Hirokazu (Katano,
JP), Kamei; Akihito (Hirakata, JP),
Kobayashi; Yasushi (Osazaki, JP), Taketani;
Makoto (Irvine, CA), Mitsumata; Tadayasu (Hirakata,
JP) |
Assignee: |
Matsushita Electric Industrial Co.,
Ltd. (Osaka, JP)
|
Family
ID: |
15027869 |
Appl.
No.: |
09/169,188 |
Filed: |
October 8, 1998 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
464116 |
Jun 5, 1995 |
05563067 |
Oct 8, 1996 |
|
|
Foreign Application Priority Data
|
|
|
|
|
Jun 13, 1994 [JP] |
|
|
6-130176 |
|
Current U.S.
Class: |
435/287.1;
324/447; 324/692; 356/246; 435/173.1; 435/288.7; 435/289.1;
435/368 |
Current CPC
Class: |
G01N
33/4836 (20130101); C12M 41/46 (20130101) |
Current International
Class: |
C12M
1/34 (20060101); G01N 33/487 (20060101); C12M
001/34 () |
Field of
Search: |
;435/287.1,288.7,289.1,173.1,368 ;204/403 ;356/246,56
;324/447,692 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
3634132 |
|
Apr 1987 |
|
DE |
|
0300651 |
|
Jan 1989 |
|
EP |
|
0367432 |
|
May 1990 |
|
EP |
|
0585933 |
|
Mar 1994 |
|
EP |
|
1514046 |
|
Aug 1976 |
|
GB |
|
52-31825 |
|
Mar 1977 |
|
JP |
|
55-84148 |
|
Jun 1980 |
|
JP |
|
63-84476 |
|
Apr 1988 |
|
JP |
|
3-265814 |
|
Nov 1991 |
|
JP |
|
4-204244 |
|
Jul 1992 |
|
JP |
|
06078889 |
|
Mar 1994 |
|
JP |
|
06296595 |
|
Oct 1994 |
|
JP |
|
WO 90/11371 |
|
Oct 1990 |
|
WO |
|
WO 91/17240 |
|
Nov 1991 |
|
WO |
|
WO 92/15700 |
|
Sep 1992 |
|
WO |
|
Other References
Baxter et al., "Microfabrication in silicon microphysiometry" Clin.
Chem. 40(9):1800-1804 (1994). .
Brochure for muti channel systems, data acquisition: High end tools
for multi electrode measurements-Mea 60-SYSTEM, mea 1060, multi
electrode array. .
Company brochure RS "Steckverbindungen-Labor/prufung 1-1193". .
Company brochure ARIES "Series 537 universal PLCC" and "Series 536
PLCC" (published later but relating to earlier-distributed
components). .
Company brochure 3M "Textool sockets and trays" (also relating to
an earlier distributed component). .
Eggers et al., "Electronically wired petri dish: A microfabricated
interface to the biological neuronal network" J. Vac. Sci. Technol.
B. 8(6):1392-1398 (1990). .
Gahwiler et al., "Multiple actions of acetylcholine on hippocampal
pyramidal cells in organotypic explant cultures" Neurosci.
7(5):1243-1256 (1982). .
Gonzales et al., "Cell and explant culture of olfactory
chemoreceptor cells" J. Neurosci. 14(2):77-90 (1985). .
Gross et al., "A new fixed-array multi-microelectrode system
designed for long-term monitoring of extracellular single unit
neuronal activity in vitro" Neurosci. Lett. 6:101-105 (1977). .
Gross et al., "Recording of spontaneous activity with photoetched
microelectrode surfaces from mouse spinal neurona in culture" J.
Neurosci. Meth. 5:13-22 (1982). .
Gross et al., "Long-term monitoring of spontaneous single unit
activity from neuronal monolayer networks cultured on photoetched
mutielectrode surfaces" J. Electrophysiol. Tech. 9:55-69 (1982).
.
Gross et al., "Multielectrode investigations of network properties
in neural monolayer cultures", In: Proc. of the sixth southern
biomedical engineering conference, pp212-217, Mc Gregor abd Werner,
Washington D.C., (1987). .
Gross et al., "An approach to the determination of network
properties in mammalian neuronal monolayer cultures" Proc. of the
First IEEE Conference on Synthetic Microstructures in Biological
Res., Arlie, VA., Mar. 24-26, 1986. .
Hammerle et al., "Extracellular recording in neuronal networks with
substrate integrated microelectrode arrays" Biosens. Bioelect.
9:691-696 (1994). .
Hazeki et al., "Modification by Islet-activating protein of
receptor-mediated regulation of cyclic AMP accumulation in isolated
rat heart cells" J. Biol. Chem. 256(6):2856-2862 (1981). .
Kuriyama et al., "A single chip biosensor" NEC Res. Develop., No.
78, pp 1-5, Tokyo, JP. (1985). .
Kuroda, "Adenosine/ATP receptor in nervous system and physiologic
function" Protein Nucl. Acid Enzyme 29(12):1405-1423 (1984) English
Abstract. .
Nakao et al., "Scanning-laser-beam semiconductor ph-imaging sensor"
Sensors & Actuators B 20(2/3):119-123 (1994). .
Nisch et al., "A thin film microelectrode array for monitoring
extracellular neuronal activity in vitro" Biosens. Bioelect.
9:737-741 (1994). .
Novac et al., "Recording from the Aplysia abdominal ganglion with a
planar microelectrode array" IEEE Trans. Biomed. Eng.
BME-33(2):196-202 (1986). .
Novak et al., "Multisite hippocampal slice recording and
stimulation using a 32 element microelectrode array" J. Neurosci.
Meth. 23:149-159 (1988). .
Novak et al., "A high-speed multichannel neural data acquisition
system for IBM PC compatabilities" J. Neurosci. Meth. 26:239-247
(1989). .
Suematsu et al., ".alpha. receptor" Protein Nucl. Acid Enzyme
29(12):1338-1352 (1984) English Abstract. .
Thomas et al., "A miniature microelectrode array to monitor the
bioelectric activity of cultured cells" Exptl. Cell Res. 74:61-66
(1972). .
Tubingen et al., "2nd CEC workshop on bioelectronics: Interfacing
biology with electronics" Biosens. Bioelect. 9:Preface (i) (1994).
.
Yamamoto, "In vitro synaptic activity" Protein Nucl. Acid Enzyme
22(6):502-505 (1977) English Abstract. .
Yamamoto et al., "Black widow spider venon: excitatory action on
hippochampal neurons" Brain Res. 244(2):382-386 (1982). .
Yamamoto, "Electrical activity of brain sector" Protein Nucl. Acid
Enzyme 29(12):1205-1211 (1984) English Abstract..
|
Primary Examiner: Redding; David A.
Attorney, Agent or Firm: Morrison & Foerster LLP
Parent Case Text
.Iadd.Application Ser. No. 09/169,188, filed Oct. 8, 1998, and
application Ser. No. 09/688,077, filed Oct. 13, 2000, are each
reissues of U.S. Pat. No. 5,563,067 (Application Ser. No.
08/464,116), filed Jun. 5, 1995..Iaddend.
Claims
What is claimed is:
1. A cell potential measurement apparatus for measurement of
electrical physiological characteristics of cells, comprising: (A)
an integrated cell holding instrument provided with a plurality of
microelectrodes arranged in a matrix form on the surface of a glass
plate, .Iadd.said microelectrodes each having an electrode area of
4.times.10.sup.2 .mu.m.sup.2 to 4.times.10.sup.4 .mu.m.sup.2,
.Iaddend.conductive patterns connected to the microelectrodes,
electric contact points which are connected to edge parts of these
conductive patterns, an insulation coating covering the surface of
said conductive patterns, said microelectrodes being in electrical
connection to a cell holding part which is constructed so as to
contain cells and arranged in an area including said plurality of
microelectrodes, and an electric connection means for providing an
electric signal to said microelectrodes and for leading out an
electric signal from said microelectrodes, said electric connection
means including a half-split holder which has contacts touching
said electric contact points and fixes said glass plate by holding
the plate at the top and bottom of the plate; (B) a stimulation
signal supply means to be connected .Iadd.to .Iaddend.the electric
connection means of said integrated cell holding instrument for
providing electric stimulation to said cells; .[.and.]. (C)
.Iadd.an optical observation means for observing the cells
optically, and (D) .Iaddend.a signal processing means to be
connected to the electric connection means of said integrated cell
holding instrument for processing an output signal arising from
electric physiological activities of said cells. .[.
2. The cell potential measurement apparatus as in claim 1, further
comprising an optical observation means for observing the cells
optically..].
3. The cell potential measurement apparatus as in claim 1, further
comprising a cell culturing means for maintaining an environment
for culturing cells which are placed on said integrated cell
holding instrument.
4. The cell potential measurement apparatus as in claim 3, wherein
the cell culturing means comprises a temperature adjustment means
for maintaining a constant temperature, a means for circulating a
culture solution, and a means for supplying a mixed gas of air and
carbon dioxide.
5. The cell potential measurement apparatus as in claim 1, wherein
said plurality of microelectrodes comprise 64 electrodes arranged
in 8 columns and 8 rows..[.
6. The cell potential measurement apparatus as in claim 1, wherein
said microelectrodes each have an electrode area of
4.times.10.sup.2 .mu.m.sup.2 to 4.times.10.sup.4
.mu.m.sup.2..].
7. The cell potential measurement apparatus as in claim 1, wherein
said electric connection means fixes said half-split holder, and
said apparatus further comprises a printed circuit board having an
outside connection pattern which is connected to the contacts of
said holder via a connector.
8. The cell potential measurement apparatus as in claim 1, wherein
contact resistance of said electric contact points with said
contacts and contact resistance of said contacts with said
connector are both less than 30 m ohm.
9. The cell potential measurement apparatus as in claim 1, wherein
said optical observation means comprises an optical microscope, and
an image pick-up device and an image display device connected to
the optical microscope.
10. The cell potential measurement apparatus as in claim 9, wherein
said optical observation means further comprises an image storage
device.
11. The cell potential measurement apparatus as in claim 1, wherein
said stimulation signal supply means comprises a pulse signal
generator.
12. The cell potential measurement apparatus as in claim 1, wherein
said signal processing means comprises a multi-channel amplifier
which amplifies a detection signal arising from cell activities and
a multi-channel display device which displays an amplified signal
waveform in real-time.
13. The cell potential measurement apparatus as in claim 1, further
comprising a computer which outputs said stimulation signal via a
D/A converter and receives and processes an output signal arising
from electric physiological activities of said cells via an A/D
converter, said computer controlling said optical observation means
and said cell culturing means..Iadd.
14. A method for the continuous, simultaneous measurement of
electrical physiological characteristics of at least one neural
sample, comprising the steps of: 1.) placing said at least one
neural sample on a microelectrode array of a measuring device,
which device comprises: (A) an integrated neural sample holding
instrument provided with said microelectrode array having plurality
of microelectrodes arranged in a matrix form, conductive pathways
connected to said microelectrodes, a neural sample holding part
which is constructed to contain at least one said neural sample and
an area including said plurality of microelectrodes, and said
conductive pathways for providing electric stimulation signals to
said microelectrodes and for leading out a responsive electric
signal from said microelectrodes; (B) a stimulation signal supply
connectable to each said conductive pathways for providing electric
stimulation to said neural sample; and (C) a signal processor
connectable to all of said conductive pathways of said integrated
neural sample holding instrument suitable for processing said
signals arising from electric physiological activities of said at
least one neural sample and reflecting said signals as said complex
waveforms, 2.) electrically stimulating at least one of said neural
samples to cause an electrical response stimulus, and 3.)
continuously simultaneously detecting said electrical response
stimulus output signals arising from electric physiological
activities of said neural sample and processing said signal to
reflect said electrical, physiological responsive, complex
waveform..Iaddend..Iadd.
15. The method of claim 14 further comprising the step of
maintaining an environment for culturing said neural sample on said
integrated neural sample holding instrument..Iaddend..Iadd.
16. The method of claim 15 further comprising the step of
maintaining a constant temperature, circulating a solution, and
supplying a gas supply to said neural sample holding
part..Iaddend..Iadd.
17. The method of claim 14 wherein said plurality of
microelectrodes comprise 64 electrodes arranged in eight columns
and eight rows..Iaddend..Iadd.
18. The method of claim 14 wherein said microelectrodes each have
an electrode area of 4.times.10.sup.2 .mu.m.sup.2 to
4.times.10.sup.4 .mu.m.sup.2..Iaddend..Iadd.
19. The method of claim 14 wherein said electrical stimulation
comprises a pulse signal..Iaddend..Iadd.
20. The method of claim 14 wherein said at least one neural sample
comprises a section of a nerve organ..Iaddend..Iadd.
21. The method of claim 14 wherein said at least one neural sample
comprises a nerve cell..Iaddend..Iadd.
22. A method for the measurement of electrical, physiological
response, complex waveforms in at least one neural samples,
comprising the steps of 1.) placing at least one neural sample on a
microelectrode array of a measuring device, which device comprises:
(A) an integrated, neural sample holding instrument provided with
i.) said microelectrode array comprising a plurality of
microelectrodes arranged in a matrix form, ii.) conductive pathways
connected to the microelectrodes, and said conductive pathways for
providing electric stimulation signals to said microelectrodes and
for leading out an electric signal from said microelectrodes and
iii.) a neural sample holding part which is constructed to contain
said at least one neural sample and include said plurality of
microelectrodes, (B) a signal processor connectable to said
conductive pathways of said integrated neural sample holding
instrument suitable for processing said signals arising from
electric physiological activities of said at least one neural
sample and reflecting said signals as said complex waveforms, 2.)
stimulating more than one of said neural samples to cause an
electrical response stimulus, and 3.) continuously, simultaneously
detecting and processing said electrical response stimulus to
reflect said complex waveforms..Iaddend..Iadd.
23. The method of claim 22 wherein the stimulation is an electrical
signal from a stimulation signal supply connectable to all of said
conductive pathways to said neural sample through said
microelectrodes..Iaddend..Iadd.
24. The method of claim 22 further comprising the step of
maintaining an environment for culturing said neural sample on said
integrated neural sample holding instrument..Iaddend..Iadd.
25. The method of claim 22 further comprising the step of
maintaining a constant temperature, circulating a solution, and
supplying a gas supply to said neural sample holding
part..Iaddend..Iadd.
26. The method of claim 22 wherein said plurality of
microelectrodes comprise 64 electrodes arranged in eight columns
and eight rows..Iaddend..Iadd.
27. The method of claim 22 wherein said microelectrodes each have
an electrode area of 4.times.10.sup.2 .mu.m.sup.2 to
4.times.10.sup.4 .mu.m.sup.2..Iaddend..Iadd.
28. The method of claim 23 wherein said electrical stimulation
comprises a pulse signal..Iaddend..Iadd.
29. The method of claim 22 wherein said said at least neural sample
comprises a section of a nerve organ..Iaddend..Iadd.
30. The method of claim 22 wherein said said at least neural sample
comprises a nerve cell..Iaddend.
Description
FIELD OF THE INVENTION
This invention relates to a cell potential measurement apparatus
which is used in the field of electrical neurophysiology for
measuring potential change associated with activities of nerve
cells or nerve organs.
BACKGROUND OF THE INVENTION
Recently, medical investigations into nerve cells and the
possibility of using nerve cells as electric elements have been
actively pursued. When nerve cells are active, action potential is
generated. This action potential rises from a change in ion
concentration inside and outside the cell membrane which is
accompanied by a change in ion permeability in nerve cells and thus
from the change in cell membrane potential accompanied thereby.
Therefore, measuring this potential change accompanied by the ion
concentration change (that is, the ion current) near the nerve
cells with electrodes enables the detection of activities of nerve
cells or nerve organs.
In order to measure the above-mentioned potential arising from cell
activities, it is possible, for example, to insert an electrode
comprising glass into an area of cells to measure extracellular
potential. When evoked potential due to stimulation is measured, a
metal electrode for stimulation is inserted together with a glass
electrode for recording. However, measurement by the insertion of
these electrodes has the possibility of damaging the cells, and
measurement over a long period of time is difficult to carry out.
In addition, due to restrictions of space and the need for
operating accuracy, multipoint simultaneous measurements are also
difficult to carry out.
Therefore, the present inventors developed a planar electrode
comprising an insulation substrate and a multiplicity of
microelectrodes and their drawer patterns formed thereon with the
use of a conductive material, and cell culture could take place on
that surface (disclosed in Laid-open Japanese patent application
Nos. (Tokkai Hei) 6-78889 and 6-296595). With this planar
electrode, multi-point simultaneous measurements of potential
change can be carried out without being affected by restrictions of
space at a plurality of points with a short electrode-to-electrode
distance. Also, this electrode enabled long-term measurement.
However, a measurement apparatus which can efficiently use this
kind of planar electrode, conduct measurements accurately and
efficiently, and improve the arranging of measurement results has
been strongly desired. Therefore, it is an object of this invention
to provide a cell potential measurement apparatus which is capable
of accomplishing these needs in the art.
SUMMARY OF THE INVENTION
In order to accomplish these and other objects and advantages, a
cell potential measurement apparatus of this invention comprises
(A) an integrated cell holding instrument provided with a plurality
of microelectrodes on a substrate, a cell holding part for placing
cells thereon, and an electric connection means for providing an
electric signal to the microelectrodes and for leading out an
electric signal from the microelectrodes; (B) a stimulation signal
supply means to be connected to the electric connection means of
the cell holding instrument for providing electric stimulation to
the cells; and (C) a signal processing means to be connected to the
electric connection means of the cell holding instrument for
processing an output signal arising from electric physiological
activities of the cells.
It is preferable that the cell potential measurement apparatus of
this invention further comprises an optical observation means for
observing the cells optically. It is also preferable that the cell
potential measurement apparatus of this invention further comprises
a cell culturing means for maintaining an environment for culture
of cells which are placed on the integrated cell holding
instrument. This configuration enables measurement over a long
period of time.
Generally, the measurement conducted by means of the
above-configured apparatus of this invention is carried out, for
example, in the following steps. Sample cells are placed in a cell
holding part of an integrated cell holding instrument, and a
plurality of microelectrodes contact the cells.
An image of the cells is obtained by an optical observation means.
A stimulation signal is applied between a pair of electrodes
selected optionally from the plurality of microelectrodes by a
stimulation signal supply means via an electric connection means. A
change of evoked potential over time which is obtained in each of
the other electrodes is provided to a signal processing means via
the electric connection means, which is then output, for example,
to a display device etc. after going through the necessary signal
processing. The measurement of spontaneous potential which is not
provided with a stimulation signal is carried out in a similar
way.
The above-mentioned electric chemical measurement of cells must be
conducted in a condition in which the cells are alive. Therefore,
it is common to use cultured cells, and the cell holding part of
the integrated cell holding instrument can be equipped with a
culture medium. Since the integrated cell holding instrument is
detachable from the measurement apparatus, each integrated cell
holding instrument can be placed inside an ordinary incubator for
cell culture and then taken out from the incubator and placed in
the measurement apparatus. When a cell culturing means is further
provided to maintain an environment for culture of the cells on the
integrated cell holding instrument, long-term measurement is
enabled. This cell culturing means comprises a temperature
adjustment means for maintaining a constant temperature, a means
for circulating a culture solution, and a means for supplying a
mixed gas of air and carbon dioxide (e.g., CO.sub.2 5%).
It is preferable that the integrated cell holding instrument
comprises a plurality of microelectrodes arranged in a matrix form
(latticed) on the surface of a glass plate, conductive patterns for
drawing these microelectrodes, electric contact points which are
connected to edge parts of these conductive patterns, and a coating
of insulation covering the surface of these conductive patterns,
and the cell holding part is disposed in an area including the
plurality of microelectrodes.
The use of a transparent glass plate as the substrate faciliates
optical observations of the cells. Therefore, it is preferable that
the conductive patterns or the insulation coating are also
substantially transparent or translucent. Furthermore, when the
plurality of microelectrodes is arranged in a matrix form, it is
easier to specify positions of electrodes which are applied with
stimulation signals or electrodes where voltage signals arising
from cell activities are detected. For example, it is preferable to
arrange 64 microelectrodes in 8 columns and 8 rows. In addition,
the surface area of each electrode should be as broad as possible
for reducing surface resistance and enhancing detection
sensitivity. However, taking restrictions etc. arising from an
electrode-to-electrode distance and space resolution of measurement
into consideration, it is preferable that each electrode has a
surface area of from 4.times.10.sup.2 .mu.m.sup.2 to
4.times.10.sup.4 .mu.m.sup.2.
Furthermore, it is preferable that the electric connection means
includes a half-split holder which has a contact touching the
electric contact point and fixes the glass plate by holding it from
the top and bottom. According to this configuration, fixation of
the glass plate and drawing of the microelectrodes to the outside
can be performed easily and accurately. Furthermore, it is
preferable that the electric connection means not only fixes the
holder, but also comprises a printed circuit board having an
outside connection pattern which is connected to the contact of the
holder via a connector. As a result, connection with outside
instruments, namely, with a stimulation signal supply means and a
signal processing means is facilitated. For the transmission of
stimulation signals or detection signals with as little attenuation
and distortion as possible, contact resistance of the electric
contact point with the contact as well as contact resistance of the
contact with the connector are both preferably below 30 m ohm.
In addition, it is preferable that the optical observation means
comprises an optical microscope, and an image pick-up device and an
image display device connected to the optical microscope. In other
words, the image of cells which is enlarged by a microscope is
picked up by an image pick-up device (e.g., video camera) and then
displayed in an image display device (e.g., a high-accuracy
display), so that it is easier to conduct measurement while
observing the cells and the electrode position. More preferably,
when the optical observation means is further comprised of an image
storage device, it is possible to record measurement results.
Also, when a pulse signal generator is used as the stimulation
signal supply means, various kinds of signal waveforms can be
applied as stimulation signals to the cells. It is preferable that
the signal processing means comprises a multi-channel amplifier
which amplifies a detection signal arising from cell activities and
a multi-channel display device which displays an amplified signal
waveform in real-time, and that signal waveforms (change of cell
potential over time) obtained from a plurality of electrodes can be
displayed simultaneously.
It is preferable that a computer is provided to output the
stimulation signal via a D/A converter, and at the same time, to
receive and process an output signal arising from electric
physiological activities of the cells via an A/D converter. As a
result, the stimulation signal can be determined as an optional
waveform on the screen or a waveform of a detection signal can be
displayed on the screen. In addition to these operations, it is
easier to display these signals after being processed in various
forms or to output them to a plotter or to store them. Furthermore,
with the use of this computer, the optical observation means and
the cell culturing means can be controlled.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view showing an integrated cell holding
instrument used for a cell potential measurement apparatus in one
embodiment of this invention.
FIG. 2 is an assembly diagram of an integrated cell holding
instrument.
FIG. 3 is a flat diagram showing 64 microelectrodes and drawer
patterns disposed on the center of a planar electrode comprising an
integrated cell holding instrument.
FIG. 4 is a model cross-sectional view of a planar electrode.
FIGS. 5(A) and 5(B) are a flat diagram and a side cross-sectional
view showing a state in which a planar electrode is fixed by being
held between upper and lower holders.
FIG. 6 is a perspective view of the planar electrode and the upper
and lower holders of FIGS. 5(A) and 5(B).
FIG. 7 is a side view of a contact equipped to an upper holder.
FIG. 8 is an assembly diagram of an integrated cell holding
instrument seen from an opposite direction of FIG. 2.
FIG. 9 is a block diagram of a cell potential measurement apparatus
in one embodiment of this invention.
FIGS. 10(A) and 10(B) are graphs showing one comparative example of
a voltage waveform arising from activities of cultured cells
measured by means of an integrated cell holding instrument used in
this invention and a voltage waveform measured by means of a
conventional general purpose glass electrode (electrode for
measurement of extracellular potential).
FIGS. 11(A) to 11(C) are diagrams showing measurement results of
spontaneous potential of cultured cells measured by using an
apparatus of this invention.
FIGS. 12(A) to 12(C) are diagrams showing measurement results of
evoked potential of cultured cells measured by using an apparatus
of this invention.
DETAILED DESCRIPTION OF THE INVENTION
This invention will now be described in detail by referring to the
attached figures and the following examples. The examples are
illustrative and should not be construed as limiting the invention
in any way.
First, an integrated cell holding instrument used for a cell
potential measurement apparatus of this invention will be
explained. The integrated cell holding instrument 1, as shown as a
perspective view in FIG. 1 and as an assembly diagram in FIG. 2,
comprises a planar electrode 2, which is disposed with a plurality
of microelectrodes and their drawer patterns on the surface of a
glass plate, half-split holders 3, 4 for fixing the planar
electrode 2 by holding it from the top and bottom, and a printed
circuit board 5 on which these holders are fixed.
The planar electrode 2 is approximately the same as that disclosed
in Laid-open Japanese patent application No. (Tokkai Hei) 6-78889
and others. The planar electrode 2 comprises, for example, a
substrate made of a transparent pilex glass having a thickness of
1.1 mm and a size of 50.times.50 mm, and in the center of this
substrate, 64 pieces of microelectrodes 11 are formed in a matrix
form of 8.times.8, and each microelectrode is connected to a drawer
conductive pattern 12 (cf. FIG. 3). Each of the electrodes 11 has a
size of 50.times.50 .mu.m square (area 25.times.10.sup.2
.mu.m.sup.2), and the center-to-center distance between the
adjacent electrodes is 150 .mu.m. Furthermore, each side of the
substrate has 16 pieces of electric contact points 7 formed,
totalling to 64 pieces (cf. FIG. 2). These electric contact points
7 are connected with 64 pieces of the microelectrodes 11 disposed
in the center of the substrate to correspond by 1 to 1 by the
drawer conductive patterns 12. 16 pieces of the electric contact
points are arranged on each side with a pitch of 1.27 mm. Next, a
method of manufacturing this planar electrode 2 will be explained
based on its cross-sectional view shown as FIG. 4. Each part in
FIG. 4 is shown on a reduced scale for convenience.
ITO (indium tin oxide), for example, was applied to form a layer of
150 nm thick on the surface of a glass plate 13, which is then
formed into the conductive pattern 12 through a photoresist and
etching. On top of this layer, a negative photosensitive polyimide
is applied to form a layer of 1.4 .mu.m thick, which is then formed
into an insulation film 14 in a similar manner. The ITO layer is
exposed at the microelectrode part, and at the part of the electric
contact point, nickel 15 of 500 nm thick and gold 16 of 50 nm
thick, are coated on these parts. A cylindrical polystyrene frame 6
(cf. FIG. 2) with an inner diameter 22 mm, an outer diameter 26 mm,
and a height 8 mm is adhered (via a conductive pattern 8 and an
insulation film 9) on the glass plate 13 using a silicone adhesive.
This cylindrical polystyrene frame 6 is fixed with its center
matching the center of the glass plate 13, that is, the central
part of 64 microelectrodes, and the inside of the polystyrene frame
6 becomes a cell holding part. The inside of this polystyrene frame
6 is filled with solutions comprising 1 wt. % of chloroplatinic
acid, 0.01 wt. % of lead acetate, and 0.0025 wt. % of hydrochloric
acid. An electric current of 20 mA/cm.sup.2 is generated for 1
minute to deposit platinum black 11a on the surface of the gold
plating of the microelectrode part.
Next, the half-split holders 3, 4 for fixing the planar electrode 2
by holding from the top and bottom will be explained. The holders
3, 4 made, for example, of resin are provided with a stage part for
holding a frame part of the planar electrode 2 and with a
rectangular opening in the central part, as shown in FIG. 2. The
upper holder 3 is equipped with a pair of fixtures 8 and 16.times.4
pairs of contacts 9.
A top face view of the holders 3, 4 which hold and fix the planar
electrode 2 is shown in FIG. 5(A), and its side view (5(B)--5(B)
cross-sectional view) is shown in FIG. 5(B), and its perspective
view seen from a bottom side is shown in FIG. 6. As clearly shown
in these figures, the fixture 8 is pivoted on two opposing sides of
the upper holder 3 by an axis pin 8a. Furthermore, a groove 4a is
formed on two opposing sides of the lower holder 4 in the bottom
face. By fitting a convex part 8b of the fixture 8 into the groove
4a, the upper and the lower holders 3, 4 are firmly fixed with the
planar electrode 2 held in between.
64 pieces of the contacts 9, which are disposed in the upper holder
3 to correspond to the electric contact points 7 of the planar
electrode 2, are formed by processing an elastic, conducting metal
plate such as a plate comprising BeCu coated with Ni and Au, and
the contact 9 has a shape shown in FIG. 7. In other words, the
contact 9 is comprised of a pin part 9a, and its base part 9b, and
a movable contact part 9d extending from the base part 9b via a
curved part 9c. According to this structure, the movable contact
part 9d is capable of elastic displacement against the base part
9b. The upper holder 3 has 64 (16.times.4) pieces of holes formed
which are inserted with the pin part 9a of the contact 9, and the
same number of grooves are also formed which fit the base part
9b.
As shown in FIG. 2 and FIG. 5(B), the pin part 9a protrudes from
the upper holder 3 at the point where the contact 9 is inserted
into the above-mentioned hole and the groove and fixed. By
alternately arranging the contact 9 having two different lengths of
the base part 9b, 16 pieces of the pin part 9a protruding from the
upper holder 3 are lined in two staggered rows. This pin part 9a is
connected to a connector which is mounted on a printed circuit
board 5 used for connection with the outside.
On the other hand, the movable contact part 9d of the contact 9
protrudes from the bottom face of the upper holder 3 at the point
where the contact 9 is inserted into the holder and the groove of
the upper holder 3 and fixed. This arrangement is shown in FIG. 8,
which is an assembly diagram seen from the side opposite the
assembly diagram of FIG. 2. In this state, the planar electrode 2
is fixed between the holders 3, 4, and the movable contact part 9d
of each contact 9 touches the electric contact point 7 of the
planar electrode 2, and a predetermined contact pressure is exerted
on the contact part due to elastic deformation of the curve part
9c. In this way, the electric contact point 7, which is connected
to the microelectrode 11 of the planar electrode 2 by way of the
conductive pattern 12, is electrically connected with small contact
resistance (less than 30 m ohm) against the contact 9.
Next, the printed circuit board 5 will be explained. This printed
circuit board 5 serves not only for fixing the assemblies of the
planar electrode 2 and the holders 3, 4, but also for drawing an
electrical connection via a connector to the outside, starting from
the microelectrode 11 of the planar electrode 2 via the conductive
pattern 12 via the electric contact point 7 to the contact 9.
Furthermore, this printed circuit board 5 facilitates handling
procedures, for example, installation to the measurement
apparatus.
This printed circuit board 5 comprises a glass epoxy substrate
disposed with double-faced patterns, and on the back face shown in
FIG. 8, a connector 5a is disposed at four parts surrounding a
circular opening formed in the center. By inserting 16 pieces of
the pin part 9a which are protruding in two staggered rows from the
four surface parts of the upper holder 3 into each corresponding
connector 5a, the assemblies of the planar electrode 2 and the
holders 3, 4 are fixed at the printed circuit board 5, and at the
same time, they are connected electrically.
At an edge part 5b on both sides of the printed circuit board 5,
electric contact points are formed at 2.54 mm pitch used for a
double-faced connector edge, and these electric contact points and
the connectors 5a in the central part are connected by a drawer
pattern 5c. An inner row of the double-sided connector 5a is drawn
by a surface pattern, whereas an outer row is drawn by a back side
pattern, and each of the edge part 5b is provided with 32 electric
contact points formed for both sides together, totalling 64
electric contact points. For the purpose of assuring mechanical
fixation, the upper holder 3 can be fixed to the printed circuit
board 5 using a vise.
A preferable configuration of a cell potential measurement
apparatus using the above-configured integrated cell holding
instrument 1 is shown in FIG. 9. The measurement apparatus of this
embodiment comprises the above-mentioned integrated cell holding
instrument 1, an optical observation means 20 including an inverted
microscope 21 for optical observations of cells which are placed in
this integrated cell holding instrument 1, a computer 30 including
a means of providing a stimulation signal to the cells and a means
of processing an output signal from the cells, and a cell culturing
means 40 for maintaining a suitable culture medium for the
cells.
Besides the inverted microscope 21 (for example, "IMT-2-F" or
"IX70" manufactured by OLYMPUS OPTICAL CO., LTD.) where the
integrated cell holding instrument 1 is installed, the optical
observation means 20 also includes a SIT camera 22 used for a
microscope (for example, "C2400-08" manufactured by HAMAMATSU
PHOTONICS K.K.), a high-accurate display 23, and an image filing
device 24 (for example, "TQ-2600" or "FTQ-3100" manufactured by
MATSUSHITA ELECTRIC INDUSTRIAL CO., LTD.). A SIT camera is a
general term used for cameras which apply a static induction
transistor to an image pickup tube, and a SIT camera is a
representative example of sensitive cameras. However, the
high-accuracy display 23 can be used also as a display for the
computer 30. The specific devices described above in parenthesis
are illustrative examples, and the invention is not limited to
these devices only. This is also the same with the examples shown
in the following.
As for the computer 30, a personal computer (for example,
compatible with WINDOWS) is used which is mounted with an A/D
conversion board and software for measurement. The A/D conversion
board includes an A/D converter 31 and a D/A converter 32 shown in
FIG. 9. The A/D converter 31 has 16 bits and 64 channels, and the
D/A converter 32 has 16 bits and 8 channels.
The measuring software includes software for determining conditions
needed for providing a stimulation signal or recording conditions
of an obtained detection signal. With the use of this type of
software, the computer 30 is not only capable of structuring the
means of providing a stimulation signal to the cells and the means
of processing the detection signal from the cells, but also is
capable of controlling the optical observation means (the SIT
camera and the image filing device) or the cell culturing
means.
In the following, particularly useful specifications for the
software for measurement will be explained. On a computer screen
directed to parameter setting, it is possible to determine
complicated stimulation conditions by drawing a stimulation
waveform on the screen using a keyboard or a mouse. Furthermore,
recording conditions are determined such that 64 input channels, a
sampling rate of 10 kHz, and continuous recording over several
hours are enabled. In addition, the electrode which provides a
stimulation signal or the electrode which draws out a detection
signal from the cells can be specified by pointing to a microscope
image displayed on the screen with a mouse or a pen. Besides,
various conditions such as temperature or pH of the cell culturing
means 40 can be determined by using a keyboard.
A recording screen displays a spontaneous action potential or an
evoked potential detected from the cells in real-time at a maximum
of 64 channels. Furthermore, the recorded spontaneous action
potential or the evoked potential can be displayed on top of a
microscope image of cells. When the evoked potential is measured,
the whole recording waveform is displayed. When the spontaneous
action potential is measured, the recording waveform is displayed
only when an occurrence of spontaneous action is detected by a
spike detection function using a window discriminator or a waveform
discriminator. When the recording waveform is displayed,
measurement parameters (e.g., stimulation conditions, recording
conditions, temperature, pH) at the time of recording are
simultaneously displayed in real-time. There is also an alarm
function provided in case when a temperature or pH goes beyond
permissive limits.
On a computer screen for data analysis, FFT analysis, coherence
analysis, and correlation analysis can be conducted. In addition,
this screen has other functions, such as a single spike separation
function using a waveform discriminator, a temporal profile display
function, a topography display function, an electric current source
density analysis function. Results of these analyses can be
displayed on top of the microscope image stored in the image filing
device.
When a stimulation signal is output from the above-configured
computer 30, this stimulation signal is forwarded by way of the D/A
converter 32 and an isolator 33 (for example, "BSI-2" manufactured
by BAK ELECTRONICS CO., LTD.) to the cells. In other words, the
stimulation signal is applied between two points selected from 64
pieces of the microelectrodes 11 in the integrated cell holding
instrument 1. Then, an evoked potential arising between each of the
microelectrodes 11 and a GND level (potential of culture solution)
is input to the computer 30 via 64 channels of a sensitized
amplifier 34 (for example, "AB-610J" manufactured by NIHON KODEN
CO., LTD.) and the A/D converter 31. The amplification factor of
the amplifier 34 was 100 dB, and the frequency band was from 0 to
10 kHz. However, when an evoked potential by a stimulation signal
is measured, the frequency band was determined to be from 100 Hz to
10 kHz using a low cut filter.
Next, the cell culturing means 40 is provided with a temperature
adjuster 41, a circulation means of culture solution 42, and a
means for supplying a mixed gas of air and carbon dioxide 43.
Actually, the cell culturing means 40 can be comprised of a product
equivalent to a microincubator such as "PDMI-2" and a product
equivalent to a temperature controller such as "TC-202" (both
products manufactured by MEDICAL SYSTEMS CO., LTD.), and a CO.sub.2
bomb, for example, is used. This microincubator can control the
temperature in the range of 0.degree. to 50.degree. C. by a Peltier
element, and this microincubator is capable of handing a liquid
delivery speed of below 3.0 ml/min and an air supply speed of below
1.01/min. Alternatively, a microincubator integrated with a
temperature controller (for example, "IMT2IBSV" manufactured by
OLYMPUS OPTICAL CO., LTD.) may be used.
A preferable embodiment of the cell potential measurement apparatus
of this invention was explained above. However, the cell potential
measurement apparatus of this invention is not limited to this
embodiment only and can be performed, for example, in various other
forms described in the following.
Although a means for providing a stimulation signal to cells was
comprised of a computer and a D/A converter in the above-mentioned
embodiment, this means may be comprised of a general purpose or a
special purpose pulse signal generator. Here, the stimulation
signal is preferably determined as a bipolar constant voltage pulse
comprising a pair of positive and negative pulses for eliminating
artifact, that is, for preventing DC components from flowing. In
addition, it is preferable to convert it to a constant electric
current pulse for preventing the electric current from flowing
excessively. For example, the stimulation signal is preferably
comprised of a positive pulse with a pulse width of 100 .mu.sec, an
interval of 100 .mu.sec, and a negative pulse of 100 .mu.sec, and
it is preferable that the peak electric current of the
positive-negative pulse is in the range of 30 to 200 .mu.A.
Furthermore, the installation of the cell culturing means 40 in the
measurement apparatus enables continuous measurement over a long
period of time. Alternatively, it is also possible to configure the
apparatus such that sample cells are placed in an integrated cell
holding instrument and cultured inside an incubator which is
prepared separately from the measurement apparatus, and such that
the integrated cell holding instrument is taken out only for a
comparatively short-term measurement from the incubator to be
installed in the measuring apparatus. In this case, the cell
culturing means 40 is not necessarily provided in the measurement
apparatus.
By using the above-mentioned cell potential measurement apparatus,
nerve cells or organs were actually cultured on the integrated cell
holding instrument and the potential change accompanied by
activities of the nerve cells or nerve organs were measured. An
example of this measurement will be explained hereafter. A cerebral
cortex section of rats were used as the nerve organs, which were
cultured according to a method which will be described later on in
an embodiment.
It will be first referred to results of comparing a voltage
waveform measured by means of an integrated cell holding instrument
of this invention and a voltage waveform measured by means of a
conventional general purpose glass electrode (electrode used for
measurement of extracellular potential). Nerve organs which were
cultured for 14 days were used as the sample. A stimulation signal
was applied between two adjacent electrodes of a planar electrode
comprising the integrated cell holding instrument, and a waveform
of evoked potential change over time which was induced at 8
electrodes close to the two electrodes was measured. For the
purpose of comparison, glass electrodes were sequentially
transferred to the vicinity of the above-mentioned eight electrodes
by using a three-dimensional micromanipulator, and the same voltage
waveform was measured.
As a result of comparing the voltage waveform measured by using a
planar electrode (integrated cell holding instrument) and the
waveform measured by using the glass electrode at eight parts, it
was clear that both waveforms were very similar at all the parts.
Representative examples of these waveforms are shown in FIG. 10(A)
and FIG. 10(B). FIG. 10(A) shows a waveform measured by a planar
electrode, and FIG. 10(B) shows a waveform measured by a glass
electrode. When both waveforms are compared, it is clear that there
is a slight difference in frequency characteristics. Compared with
the measurement using a planar electrode, the measurement using a
glass electrode shows a small damage sustained to the follow-up
property toward a rapid potential change. This is considered to
result from a capacitance difference of a glass electrode and a
planar electrode.
Next, an experiment was conducted to examine the relationship
between progressive days of nerve organs cultured on an integrated
cell holding instrument and the potential distribution arising from
cell activities. Prior to culture of the cells, the surface of a
planar electrode was covered with collagen gel for the purpose of
enhancing the adhesive property of each electrode in the planar
electrode with the cells. In other words, collagen gel with a
thickness of less than 50 .mu.m was formed on the surface of each
electrode coated with platinum black and also on the surface of an
insulation coating in the vicinity thereof as mentioned above.
Then, on top of the collagen gel, and also where a microelectrode
is present, a section of cerebral cortex of rats (thickness of less
than 500 .mu.m) was placed and cultured. Measurement results of the
spontaneous potential are shown in FIGS. 11(A) to 11(C), and
measurement results of the evoked potential at the time when a
stimulation signal is provided are shown in FIG. 12.
FIG. 11(A) shows a microscopic image of the sample cells and the
microelectrodes, and waveforms of the spontaneous potential
measured at seven electrode parts indicated as 1 to 7 on this image
are shown in FIG. 1(B) and FIG. 1(C). FIG. 11(B) is a waveform
measured on the sixth day after culture, and FIG. 11(C) is a
waveform measured on the tenth day after culture. A scale of the
microscopic image, time of the measurement waveforms, and a scale
of the voltage are indicated in the figure. According to the
measurement results, it is confirmed, for example, that on the
sixth day after culture, the spontaneous activities of the cells
measured at each electrode are weak, and synchronic property of
electrodes to each other can be hardly observed, whereas on the
tenth day after culture, a large number of nerve cells become
active simultaneously, indicating that the synchronic property of
electrodes to each other increased.
FIG. 12(A) also shows a microscopic view of the sample cells and
the microelectrodes. Image processing, which is included in the
software for measurement in the above-mentioned computer, was
applied to draw an outline of the cells and positions of each
electrode from the microscopic image onto the screen. Furthermore,
the voltage waveform measured at each electrode was displayed
thereon, as shown in FIG. 12(B) and FIG. 12(C). FIG. 12(B) shows a
distribution of the evoked potential on the fifth day after
culture, and FIG. 12(C) shows the same on the tenth day after
culture. A pair of electrodes indicated on the upper right side
with a + and - sign are electrodes applied with a stimulation
signal. Right above a small square sign showing the position of
each electrode, a waveform measured by this electrode is displayed.
In these waveforms, a part where a large vertical swing is observed
on the left end is an artifact corresponding directly to the
stimulation signal, and the potential change after the artifact
indicates actual cell activities. As a result of these
measurements, it is confirmed, for example, that on the fifth day
after culture, the cell activities are limited in a place which is
comparatively close to the electrode positions applied with the
stimulation signal, but on the tenth day after culture, the cell
activities can be observed in a wide range and their scale
(amplitude) becomes larger.
Next, examples of a suitable culture method for cerebral cortex
slices will be explained.
1) Culture medium
The following additives were added to a culture medium in which
Dulbecco modified Eagle's medium and HamF-12 medium were mixed in a
volume ratio of 1:1 (media manufactured by GIBCO CO., LTD.
430-2500EB). * glucose, GIBCO CO., LTD. 820-5023IN, 2.85 mg/L
(totalling to 6 mg/L together with glucose contained originally in
the above-mentioned culture medium) * putrescine, SIGMA CO., LTD.
P5780, 100 .mu.M * progesterone, SIGMA CO., LTD. P8783, 20 nM *
hydrocortisone, SIGMA CO., LTD. H0888, 20 nM * sodium selenite,
WAKO CO., LTD. 198-0319, 20 nM * insulin, SIGMA CO., LTD. 16634, 5
mg/L * transferrin, SIGMA CO., LTD. T147, 100 mg/L * sodium
bicarbonate, CO., LTD. 2.438 g/L * addition of a suitable amount of
1N HCl or 1N NaOH to adjust to pH 7.4
After the above-mentioned additives were added, filtration and
sterilization were conducted, and the culture medium was perserved
at 4.degree. C. and ready to be used. This culture medium is
hereinafter simply called "culture medium"
2) Structure of a well on a planar electrode
For the convenience of culturing nerve cells or nerve organs on a
planar electrode, a polystyrene cylinder having an inner diameter
22 mm, an outer diameter 26 mm, and a height 8 mm was adhered in
the following steps.
(a) On the bottom face of a polystyrene cylinder (inner diameter 22
mm, outer diameter 26 mm, height 8 mm), a sufficient amount of an
one-liquid silicon adhesive (DOW CORNING CO., LTD. 891 or SHIN-ETSU
CHEMICAL CO., LTD. KE-42RTV) was applied.
(b) The center of a glass substrate in the planar electrode and the
center of the polystyrene cylinder were carefully matched and then
adhered in this state.
(c) By leaving it in an environment in which dust hardly enters for
24 hours, the adhesive was solidified.
(d) After dipping in 70% ethanol for 5 minutes, sterilization was
conducted by air-drying inside a clean bench, which is then ready
for processing the electrode surface.
3) Processing of the electrode surface
In order to enhance cell adhesive property on the surface of a
planar electrode, collagen gel was formed on the surface of the
electrode by the following method. All of these operations were
conducted under a sterilized atmosphere.
(a) Solutions A, B, and C were prepared and iced.
A 0.3 vol. % diluted hydrochloric acid collagen solution (pH 3.0,
NITTA GELATIN CO., LTD. Cellmatrix Type I-A)
B. Solution comprising a mixture medium of Dulbecco modified
Eagle's medium and HamF-12 medium mixed in a volume ratio of 1:1
(GIBCO CO., LTD. 430-2500EB), which is not provided with sodium
bicarbonate and is made with a concentration 10 times higher than
for an ordinary use, and then filtration and sterilization were
conducted thereto.
C. 2.2 g of sodium bicarbonate and 4.77 g of HEPES (manufactured by
GIBCO CO., LTD. 845-1344 IM) were dissolved in 100 mL of 0.05N
sodium hydroxide solution, and filtration and sterilization were
conducted thereto.
(b) While cooling, the solutions A, B, and C were mixed at a volumn
ratio of 8:1:1:. At this time, A and B are first mixed thoroughly
and C is added afterwards to be mixed.
(c) In a well of a planar electrode which was cooled in advance to
about 4.degree. C., 1 mL of the mixed solution of (b) was injected
little by little. After the entire electrode surface was covered,
the mixed solution was removed as much as possible with a glass
Pasteur pipette. Through this operation, a coating of the mixed
solution was formed on the electrode surface with a thickness of
less than 50 .mu.m.
(d) By heating the planar electrode disposed with the mixed
solution coating at 37.degree. C. for 30 minutes, gelatinization of
the mixed solution took place, and a collagen gel matrix was
formed.
(e) 1 mL of sterilized water was added into the well of the planar
electrode, and about 5 minutes thereafter, the water was removed,
thereby washing.
(f) The operation of Step (e) was repeated two more times (a total
of 3 times).
(g) 1 mL of the culure medium (excluding insulin and transferrin)
was injected little by little into the well of the planar
electrode, and preserved inside a CO.sub.2 incubator under the
conditions of temperature 37.degree. C., relative humidity 97% and
higher, CO.sub.2 concentration 5%, and air concentration 95%, which
is then ready for use.
4) Culture of nerve cells or nerve organs
Generally speaking, culture forms can be divided into two types.
That is, a dissociated cell culture of nerve cells and an
organotypic slice culture of a nerve organ. Each form will be
explained in the following.
4-1) Dissociated culture of cerebral visual cortex nerve cells of
rats
The following operations were all performed in a sterilized
atmosphere.
(a) Brains of fetuses of SD rats at 16-18 days of pregnancy were
removed and immersed in iced Hanks' Balanced Salt Solution
(manufactured by GIBCO CO., LTD. 450-1250EB).
(b) From the brains in the iced Hanks' Balanced Salt Solution,
visual cortices were cut out and transferred to minimum essential
medium liquid (manufactured by GIBCO CO., LTD. 410-1100EB).
(c) In the minimum essential medium liquid, the visual cortices
were cut into as small pieces as possible, 0.2 mm square at
maximum.
(d) The visual cortices cut into small pieces were placed in test
tubes for centrifugal separation, and after washing with Hanks'
Balanced Salt Solution free from calcium and magnesium three times,
they were dispersed in a suitable volume of the same liquid.
(e) In the test tubes for centrifugal separation of Step (d),
Hanks' Balanced Salt Solution free from calcium and magnesium with
trypsin dissolved at 0.25% was added to double the total volume.
With gentle stirring, enzymatic processes were allowed to take
place while the solution was constantly kept at 37.degree. C. for
15 minutes.
(f) To the culture medium shown in 1) (containing additives,
hereinafter abbreviated as a culture medium), 10 vol. % of fetal
cow serum was added, which is then placed in the test tubes for
centrifugal separation subjected to Step (e) to further double the
total volume. With a glass Pasteur pipette having a reduced
diameter produced by fire-polishing the tip end with a burner,
gently repeating piperting (about 20 times at maximum), the cells
were unravelled.
(g) Centrifugation was carried out for 5 minutes at 9806.65
m/sec.sup.2 (that is, 1000 g). Upon completion of centrifugation,
the supernatant was discarded and the precipitate was suspended in
the culture medium containing 5 vol. % of fetal cow serum.
(h) Step (g) was repeated two more times (a total of 3 times).
(i) The precipitate finally obtained was suspended in the culture
medium containing 5 vol. % fetal cow serum, and using an
erythrocytometer, the cell concentration in the suspension liquid
was measured. After the measurement, using the similar culture
medium, the cell concentration was adjusted to be 2 to
4.times.10.sup.6 cells/mi.
(j) A planar electrode which was preserved in a CO.sub.2 incubator
after subjected to the process of above steps 1-3) was taken out,
and the culture medium (free from insulin and transferrin) inside a
well is removed, and 500 .mu.L of a culture medium containing 5% of
fetal cow serum was newly injected little by little. Furthermore,
100 .mu.L of the cell suspension liquid with the cell concentration
adjusted according to Step (i) was gently added and again let stand
in the CO.sub.2 incubator.
(k) Three days after the performance of Step (j), one half the
culture medium was replaced with a new one. For the replaced
medium, the culture medium not containing fetal cow serum was used.
By reducing the concentration of fetal cow serum, growth of cells
other than nerve cells (for example, glial cells) can be
suppressed.
(1) Thereafter, half of the medium was replaced in a similar manner
every 1 to 2 days.
4-2) Culture method of a cerebral cortex section of rats
(a) Brains of SD rats 2 days old were removed and immersed in iced
Hanks' Balanced Salt Solution containing 0.25 vol. % of
D-glucose.
(b) In the iced Hanks' Balanced Salt Solution containing 0.25 vol.
% of D-glucose, cerebral meninges attached on the brain are removed
using a sharp-edged pincette very carefully not to damage the
cerebral cortex.
(c) About 500 .mu.m away from a callous body, a hemisphere of the
cerebral cortex without the cerebral meninges was cut from the
occipital lobe side to the frontal lobe side along the callous body
by means of microscissors used for surgical operations of eyes.
(d) Subsequently, using the microscissors used for surgical
operations of eyes, a cerebral cortex was cut out vertically to the
cross-section of Step (c) with a thickness of 200 to 300 .mu.m to
create a section.
(e) The microscissors used for surgical operations of eyes are used
further to adjust a size of the section to be about 1.times.1
mm.
(f) The planar electrode prepared in the above-mentioned "3)
Processing of an electrode surface" was taken out from the CO.sub.2
incubator, and the cerebral cortex section whose size was adjusted
was sucked up with a pipette having a diameter of 2 mm and larger
very gently not to damage the section, and then transferred into a
culture well of the planar electrode.
(g) With a Pasteur pipette with the tip end fire-polished with a
burner, the material was arranged on the electrode such that the
layer structure of the cortex faces upward and is placed on the
electrode, while being careful not to damage the cerebral cortex
section.
(h) After the cerebral cortex section is placed on the planar
electrode, the amount of the culture medium was adjusted so that a
base of the section touched the culture medium and the top face was
exposed to outside air.
(i) After adjusting the culture medium amount, the planar electrode
was placed in a sterilized Petri dish, and about 5 ml of sterilized
water at 37.degree. C. was injected little by little into the Petri
dish to prevent the culture medium from drying, and again let stand
in the CO.sub.2 incubator.
(j) Thereafter, the medium was replaced with a new one once every
day while attending to the amount of culture medium. The culture
medium amount was determined to be the same as in Step (h).
The invention may be embodied in other forms without departing from
the spirit or essential characteristics thereof. The embodiments
disclosed in this application are to be considered in all respects
as illustrative and not as restrictive. The scope of the invention
is indicated by the appended claims rather than by the foregoing
description, and all changes which come within the meaning and
range of equivalency of the claims are intended to be embraced
therein.
* * * * *