U.S. patent application number 12/964094 was filed with the patent office on 2011-11-10 for device for measuring activity of cultured cells, microchamber and method of measuring activity of cultured cells.
This patent application is currently assigned to NIKON CORPORATION. Invention is credited to Takanori ICHIKI, Hirofumi SHIONO.
Application Number | 20110275106 12/964094 |
Document ID | / |
Family ID | 41444283 |
Filed Date | 2011-11-10 |
United States Patent
Application |
20110275106 |
Kind Code |
A1 |
ICHIKI; Takanori ; et
al. |
November 10, 2011 |
DEVICE FOR MEASURING ACTIVITY OF CULTURED CELLS, MICROCHAMBER AND
METHOD OF MEASURING ACTIVITY OF CULTURED CELLS
Abstract
A device for measuring activity of cultured cells includes a
position detecting unit specifying a position of a cell to be
measured, a microchamber controlling unit disposing in the culture
container a microchamber which surrounds the cell and forms a
measurement space, the measurement space being minute with respect
to a volume of the culture container, and a measuring unit
measuring environmental factors contained in the measurement
space.
Inventors: |
ICHIKI; Takanori;
(Bunkyo-ku, JP) ; SHIONO; Hirofumi; (Fujisawa-shi,
JP) |
Assignee: |
NIKON CORPORATION
Tokyo
JP
THE UNIVERSITY OF TOKYO
Tokyo
JP
|
Family ID: |
41444283 |
Appl. No.: |
12/964094 |
Filed: |
December 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/JP2009/002952 |
Jun 26, 2009 |
|
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12964094 |
|
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Current U.S.
Class: |
435/29 ;
435/287.1; 435/288.7 |
Current CPC
Class: |
G01N 33/5008 20130101;
C12Q 1/02 20130101; C12M 41/46 20130101 |
Class at
Publication: |
435/29 ;
435/287.1; 435/288.7 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12M 1/34 20060101 C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 26, 2008 |
JP |
2008-167435 |
Claims
1. A device for measuring activity of cultured cells, the device
comprising: a position detecting unit specifying a position of a
cell to be measured existing in a culture container; a microchamber
controlling unit disposing in the culture container a microchamber
which surrounds the cell to be measured and forms a measurement
space, the measurement space being minute with respect to a volume
of the culture container; and a measuring unit measuring
environmental factors contained in the measurement space.
2. The device for measuring activity of cultured cells according to
claim 1, further comprising: an imaging device imaging the cell to
be measured in the culture container, wherein the position
detecting unit specifies the position of the cell based on an
observation image of the cell in the culture container imaged by
the imaging device.
3. The device for measuring activity of cultured cells according to
claim 1, wherein the microchamber controlling unit positions the
microchamber to the position of the cell to be measured specified
by the position detecting unit.
4. The device for measuring activity of cultured cells according to
claim 1, wherein the measuring unit performs measurement on the
cell to be measured specified by the position detecting unit.
5. The device for measuring activity of cultured cells according to
claim 1, wherein information of the environmental factors measured
by the measuring unit is correlated with each of identification
information of the cell to be measured and measurement date and
time information of the measuring unit, and is stored in a memory
unit.
6. The device for measuring activity of cultured cells according to
claim 2, further comprising: a display unit displaying the
observation image of the cell in the culture container imaged by
the imaging device; and an operation unit designating the cell to
be measured based on the observation image displayed on the display
unit.
7. The device for measuring activity of cultured cells according to
claim 1, wherein the measuring unit includes an environmental
factor sensor disposed on an inside wall of the microchamber, and
performs measurement of the environmental factors with the
environmental factor sensor.
8. The device for measuring activity of cultured cells according to
claim 1, wherein the measuring unit performs optical measurement of
the environmental factors with a microscope.
9. The device for measuring activity of cultured cells according to
claim 1, wherein: the microchamber has a cup shape having an open
bottom side to be in contact with a culture plane of the culture
container and a closed upper side, the cup shape having a diameter
of 50 mm to 100 mm and forming a measurement space; and an
environmental factor sensor measuring environmental factors is
disposed on an inside of the cup shape.
10. A microchamber having a cup shape which has an open bottom side
to be in contact with a culture plane of the culture container and
a closed upper side and forms a measurement space, comprising an
environmental factor sensor measuring environmental factors is
disposed on an inside of the cup shape.
11. The microchamber according to claim 10, wherein a diameter of
the cup shape is 50 mm to 100 mm.
12. The microchamber according to claim 10, wherein the
microchamber is formed of a material of amorphous fluororesin.
13. A method for measuring activity of cultured cells, the method
comprising: specifying a position of a cell to be measured existing
in a culture container; disposing in the culture container a
microchamber which surrounds the cell to be measured and forms a
measurement space, the measurement space being minute with respect
to a volume of the culture container; and measuring environmental
factors contained in the measurement space.
14. The method for measuring activity of cultured cells according
to claim 13, further comprising: imaging the cell to be measured in
the culture container, wherein in the specifying of the position,
the position of the cell is specified based on an observation image
of the cell in the culture container imaged by the imaging.
15. The method for measuring activity of cultured cells according
to claim 13, wherein information of the environmental factors
measured in the measuring is correlated with each of identification
information of the cell to be measured and measurement date and
time information of the measuring, and is stored in a memory
unit.
16. The method for measuring activity of cultured cells according
to claim 14, further comprising: displaying the observation image
of the cell in the culture container imaged in the imaging; and
designating the cell to be measured based on the observation image
displayed in the displaying.
17. The method for measuring activity of cultured cells according
to claim 13, wherein in the disposing of the microchamber, the
microchamber is positioned to the position of the cell to be
measured specified in the specifying of the position.
18. The method for measuring activity of cultured cells according
to claim 13, wherein in the measuring, measurement is performed on
the cell to be measured specified in the specifying of the
position.
Description
CROSS REFERENCE TO THE RELATED APPLICATIONS
[0001] This application is a continuation application of
International Application No. PCT/JP2009/002952, filed on Jun. 26,
2009, designating the U.S., in which the International Application
claims a priority date of Jun. 26, 2008, based on prior filed
Japanese Patent Application No. 2008-167435, the entire contents of
which are incorporated herein by reference.
BACKGROUND
[0002] 1. Field
[0003] The present application relates to a device for measuring
activity of cultured cells, and to peripheral technologies
thereof.
[0004] 2. Description of the Related Art
[0005] Technologies for measuring biological activity of cultured
cells are fundamental technologies in broad fields including
state-of-the-art medical field such as regenerative medicine,
screening of pharmaceutical products, and the like. For example,
there exists processes to multiply and differentiate cells in vitro
in the field of regenerative medicine. In these processes, it is
inevitable to measure biological activity of cultured cells in
order to control success/failure of differentiation of cells,
canceration of cells, and presence of infection.
[0006] On the other hand, among methods for measuring activity of
cells which have been known publicly, there is measurement with a
flow cell sorter as an example of a method for analyzing and then
separating cells. In the flow cell sorter, cells after being
subjected to fluorescence staining treatment are isolated in a
droplet which is given an electric charge, and this droplet is
dropped. Then, the direction of falling of the droplet is
controlled by applying an electric field based on the presence of
fluorescence in the cells in this droplet and the amount of
scattering light, thereby allowing collection of the cells in a
fractionating manner in plural containers (see, for example,
Kamarck, M. E., Methods Enzymol., Vol. 151, pp. 150 to 165,
(1987)).
[0007] Further, as a method for evaluating activity of a mass of
cells, there are also known enzyme immunoassay (EIA) and
fluoroimmunoassay (FIA), or ELISA combining both of them for
performing high sensitivity analysis of substances in liquid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1A, FIG. 1B are schematic views of a method for
measuring activity according to one embodiment.
[0009] FIG. 2 is a cross-sectional view schematically illustrating
a structure example of a microchamber according to one
embodiment.
[0010] FIG. 3 is a cross-sectional view schematically illustrating
another structure example of the microchamber.
[0011] FIG. 4 is a cross-sectional view schematically illustrating
another structure example of the microchamber.
[0012] FIG. 5A, FIG. 5B are views schematically illustrating a
method for measuring activity of cultured cells according to
another embodiment.
[0013] FIG. 6 is a partial cross-sectional view schematically
illustrating a microchamber sheet according to another
embodiment.
[0014] FIG. 7 is a view illustrating a correspondence between a
culture plane of a culture container and the microchamber sheet
used in another embodiment.
[0015] FIG. 8 is a block diagram illustrating a structure example
of a device for measuring activity.
[0016] FIG. 9 is a flowchart illustrating an operation example of
the device for measuring activity.
[0017] FIG. 10 is a schematic diagram illustrating the structure of
a measurement system in an example.
[0018] FIG. 11 is a graph illustrating a relation between
fluorescence intensity and a dissolved oxygen concentration in the
example.
[0019] FIG. 12 is a graph illustrating a relation between an
inverse number of the fluorescence intensity and the dissolved
oxygen concentration in the example.
[0020] FIG. 13 is a graph illustrating a calibration curve in the
example.
DETAILED DESCRIPTION OF THE EMBODIMENTS
Description of a Method for Measuring Activity of a First
Embodiment
[0021] FIG. 1 are views illustrating an overview of a method for
measuring activity of cultured cells according to one embodiment.
In one embodiment, in a culture container 13 such as a dish
containing cultured cells 12 together with a liquid medium 11, a
microchamber 14 surrounding a cell 12 to be measured is disposed,
so as to measure environmental factors included in the space in the
microchamber 14.
[0022] Here, the environmental factors in this specification mean
substances produced or consumed through the process of metabolism
in a cell. For example, the environmental factors include glucose,
calcium ions, potassium ions, sodium ions, hydrogen ions, oxygen,
reactive oxygen species, hydrogen peroxide, carbon dioxide,
proteins and peptides produced by a cell (for example, cytokine,
hormone, and the like), and so on.
[0023] FIG. 2 is a cross-sectional view schematically illustrating
a structure example of the microchamber 14 according to one
embodiment. An overall shape of the microchamber 14 of one
embodiment is formed as a bottomed cylindrical cup shape having an
open bottom side to be in contact with a culture plane of the
culture container 13 and a closed upper side. Accordingly, the cell
12 to be measured can be accommodated in the microchamber 14. In
one embodiment, by disposing the microchamber 14 on the culture
plane of the culture container 13, a measurement space which is
sufficiently small with respect to the volume of the culture
container 13 is built inside the microchamber 14. Here, the size of
the microchamber 14 is selected appropriately according to the type
and the number of cells 12 to be measured, the types of the
environmental factors to be measured, and a required time for
measurement, and so on. Here, the diameter of the chamber is set to
about 50 .mu.m to about 100 .mu.m, for example.
[0024] Further, an environmental factor sensor 15 and biomolecule
detecting probes 16 are fixed in the microchamber 14 of one
embodiment. The environmental factor sensor 15 converts the amount
of environmental factors contained in a culture solution into an
electric signal by an electrochemical method. For example, the
environmental factor sensor 15 is formed of an oxygen sensor and/or
various bio-sensors (such as glucose sensor).
[0025] The biomolecule detecting probes 16 are formed of molecules
which specifically complement target molecules selected from the
environmental factors, for which proteins such as antibodies and
enzymes, peptides, saccharides, or the like can be used. In one
embodiment, different kinds of biomolecule detecting probes 16 may
be fixed in arrays at specific positions of the microchamber 14 (on
an upper face or the like of the microchamber 14) by patterning or
the like. In such a structure, exhaustive analysis of many items at
the same time is possible. Further, when target molecules are
marked by fluorescence, or the like, coupling between the
biomolecule detecting probes 16 and the target molecules can be
measured easily. In addition, in the above-described structure, the
amount of target molecules can be measured also by fluorescence
intensity.
[0026] Here, the microchamber 14 of one embodiment may have the
following structures from (1) to (5) (or a combination of
them).
[0027] (1) When the environmental factors are measured by optical
measurement (fluorescence measurement, measurement of color
changes, absorbance measurement), it is preferred that the
microchamber 14 be formed of a translucent material passing light
having the wavelength corresponding to an optical change which
occurs in the microchamber 14. At this time, in view of
facilitating the optical measurement, it is preferred that the
refractive index of the microchamber 14 be set to the same value as
the refractive index of the liquid medium 11. Examples of materials
having a translucency to visible light and a refractive index which
is approximately equivalent to that of the liquid medium 11 include
an amorphous fluororesin (for example, Cytop (registered
trademark)), and the like.
[0028] Further, for performing the optical measurement, to avoid
distortions in an optical image in the measurement, the upper face
of the microchamber 14 may be flat. Alternatively, to improve NA in
the optical measurement, a microlens may be formed on the upper
face of the microchamber 14 (illustration of this is omitted).
[0029] (2) The microchamber 14 may be formed of an elastic material
with high flexibility. With such a structure, for example, the
microchamber 14 can be placed to cover the cell 12 to be measured
without damaging an axial fiber, or the like connected to external
cells. Further, when the microchamber 14 is slanted with respect to
the culture plane of the culture container 13, errors in alignment
can be absorbed by deformation of the microchamber 14. Here, an
example of the elastic material used for the microchamber 14 is a
silicone rubber (polydimethylsiloxane).
[0030] (3) The microchamber 14 may be formed of a material having
an oxygen permeability. With such a structure, when measurement is
performed for a relatively long time, an incubating environment of
cells can be maintained in the microchamber 14. Of course, this
structure is on the assumption that the concentration of oxygen is
not included in items of measurement. Here, an example of an oxygen
permeable material used for the microchamber 14 is a silicone
rubber (polydimethylsiloxane).
[0031] (4) To facilitate filling of the measurement space with the
liquid medium 11, hydrophilicity may be given to an inner surface
of the microchamber 14 by forming a hydrophilic film, or the like.
In this structure, when the liquid medium 11 is filled in the
container in advance with the microchamber 14 being turned upside
down so that air does not enter, and thereafter the microchamber 14
is returned to the original state (with the opening being on the
bottom side), the liquid in the container is maintained by surface
tension. Therefore, in this structure, the microchamber 14 filled
with the liquid medium 11 in advance can be placed to cover the
cells 12.
[0032] (5) On the surface of the microchamber 14, a film may be
formed of a substance (polyethylene glycol (PEG),
2-methacryloyloxyethyl phosphorylcholine (MPC), or the like) which
inhibits absorption of protein. In this structure, the influence of
measurement to the incubating environment can be lowered.
[0033] Next, procedures of the method for measuring activity of
cultured cells in one embodiment will be described
specifically.
[0034] In a first procedure, the microchamber 14 is disposed so as
to surround the cell 12 to be measured from an upper side in the
culture container 13. Thus, the cell 12 to be measured is
accommodated, and the measurement space which is minute with
respect to the volume of the culture container 13 is formed inside
the microchamber 14.
[0035] Further, to suppress adverse effects to the incubating
environment by movement of heat, it is preferred that the
microchamber 14 be kept warm at the same temperature as the medium
11. Here, there may be plural cells 12 to be accommodated in the
measurement space, but in one embodiment, one cell is accommodated
in the microchamber 14.
[0036] In a second procedure, the environmental factors contained
in the measurement space are measured. Thus, it is possible to
evaluate activity of the cell to be measured, the degree of
apoptosis or differentiation of the cell, the nature of the cell,
and so on based on the amount of the environmental factors
measured.
[0037] When the environmental factors in the culture container 13
are measured, metabolites and the like scatter in the entire
medium, and thus it is impossible to measure the environmental
factors focusing on each individual cell. On the other hand, in one
embodiment, the microchamber 14 is disposed in the culture
container 13, and the minute measurement space including the cell
12 to be measured is formed, so as to measure the environmental
factors. The environmental factors included in this measurement
space relate closely to the cell 12 to be measured, and thus it is
possible to measure local environmental factors focusing on the
cell 12 to be measured under the incubating environment.
[0038] Further, since the measurement space in the microchamber 14
is minute with respect to the volume of the culture container 13, a
change in concentration of metabolites or the like becomes large in
the measurement space as compared to the environment outside the
microchamber 14. Accordingly, the environmental factors can be
detected with quite high sensitivity. Since a change in
concentration of the environmental factors becomes large in the
measurement space, it is possible to perform measurement in a
relatively short time.
[0039] Moreover, in one embodiment, activity of the cell is
evaluated with the environmental factors included in the space
surrounding the cell 12 being the subject to be measured.
Accordingly, the influence of measurement to the cell can be
reduced as compared to when the cell itself is the subject to be
measured. Further, in one embodiment, since the cell 12 to be
measured can be measured as it is in the culture container 13, the
influence to the cell 12 and the surrounding incubating environment
by measurement can be reduced significantly.
[0040] Here, the measurement method in the second procedure will be
described in more detail. Specifically, in the second procedure,
the environmental factors in the measurement space are measured by
the environmental factor sensor 15 disposed in the microchamber 14.
Alternatively, in the second procedure, an optical change in the
measurement space caused by the biomolecule detecting probes 16
disposed in the microchamber 14 or a reagent (such as pH indicator)
may be observed with a microscope, so as to perform optical
measurement of the environmental factors (such as fluorescence
measurement, measurement of color changes, and absorbance
measurement). In addition, the biomolecule detecting probes used in
the aforementioned optical measurement may be one fixed to the
inner surface of the microchamber 14, or one dropped directly in
the medium 11 in the culture container 13 without being fixed to
the microchamber 14.
[0041] Further, in the second procedure, to allow metabolites to
accumulate sufficiently in the measurement space in the
microchamber 14, the environmental factors may be measured when a
predetermined standby time has passed after the measurement space
is formed. Furthermore, in the second procedure, the environmental
factors may be measured a plural number of times while respective
measurement periods are varied. In this way, it becomes possible to
obtain the rate of change of the amount of the environmental
factors based on changes in the amount of the environmental factors
and intervals of measurement.
[0042] In the third procedure, after the measurement of the
environmental factors is finished, the microchamber 14 is removed
from the culture container 13. Thus, the incubating environmental
of cells can be recovered to almost the same state as that before
the measurement. Therefore, in one embodiment, it is possible to
periodically determine activity of the cell 12 incubated under the
incubating environment in a substantially non-invasive manner by
repeating from the first procedure to the third procedure described
above.
[0043] In addition, in one embodiment, a plurality of microchambers
14 may be disposed in one culture container 13 to perform
measurement of activity of different cells 12 in parallel.
[0044] <Modification example of one embodiment>
[0045] FIG. 3 and FIG. 4 are cross-sectional views schematically
illustrating other structure examples of the microchamber 14 of one
embodiment. Also with these structures, a minute measurement space
can be formed surrounding the cell 12 to be measured, and
substantially the same effects as those of the microchamber 14
illustrated in FIG. 2 can be obtained. Note that in FIG. 3 and FIG.
4, components common to FIG. 2 are given the same reference
numerals, and duplicated descriptions are omitted.
[0046] The microchamber 14 illustrated in FIG. 3 is formed of a
cylindrical member in which both of a bottom face to be in contact
with the culture container 13 and an upper face are open. With the
structure in FIG. 3, air can be let out via the opening on the
upper side when the microchamber 14 is place to cover the cell 12.
In addition, in the structure of FIG. 3, the height of the
microchamber 14 needs to be set so as to prevent flowing in of the
medium 11 from the upper side of the microchamber 14.
[0047] The microchamber 14 illustrated in FIG. 4 is formed of a
cylindrical member in which both of a bottom face to be in contact
with the culture container 13 and an upper face are open. In the
microchamber 14 of FIG. 4, an opening part on an upper side is
covered with a hydrophobic semi-permeable film 17 which blocks the
medium 11 flowing in or out and allows air to flow in or out. With
the structure of FIG. 4, it is possible to let air out in one way
via the opening on the upper side when the microchamber 14 is
placed to cover the cell 12, and it is possible to prevent the
medium 11 from mixing in and out of the microchamber 14 when the
measurement space is formed.
[0048] <Description of a Method for Measuring Activity of
Another Embodiment>
[0049] FIG. 5 are views schematically illustrating a method for
measuring activity of cultured cells according to another
embodiment. Another embodiment is a modification example of one
embodiment illustrated in FIG. 1, in which measurement of the
environmental factors is performed in each of measurement spaces
using a microchamber sheet 21 in which microchambers 14 are
arranged. In addition, in another embodiment, the culture plane on
the side of the culture container 13 is modified to control
adhering positions of cultured cells 12 to be aligned with the
microchamber sheet 21.
[0050] FIG. 6 is a partial cross-sectional view schematically
illustrating the microchamber sheet 21 according to another
embodiment. Further, FIG. 7 is a view illustrating a correspondence
between the culture plane of the culture container 13 and the
microchamber sheet 21 used in another embodiment. The overall shape
of the microchamber sheet 21 according to another embodiment is a
disc-like member with an outside diameter smaller than the inside
diameter of the culture container 13, and a plurality of
microchambers 14 are formed in one face (bottom side). The
microchambers 14 are each formed of a cylindrical recessed portion
with a bottom, and are formed by a publicly known microfabrication
method, such as lithography, for example.
[0051] The respective microchambers 14 of the microchamber sheet 21
are arranged two-dimensionally at certain intervals. In FIG. 5 and
FIG. 7, for example, the microchamber sheet 21 with 4.times.4
arrays of microchambers 14 is illustrated.
[0052] On the other hand, on the culture plane of the culture
container 13, first regions 22 and a second region 23 which have
different adherences to cells are formed. The first regions 22 have
a relatively high adherence to cells on the culture plane. A
plurality of first regions 22 are formed on the culture plane, and
the number of first regions corresponds to the number of
microchambers 14 of the microchamber sheet 21. The first regions 22
on the culture plane form 4.times.4 arrays aligned with the
intervals of the microchambers 14. Here, the size of each first
region 22 is set to be slightly smaller than the size of the
microchambers 14.
[0053] The second region 23 has a relatively low adherence to cells
on the culture plane, and is formed to surround the first regions
22. Accordingly, in the culture container 13, cells adhere easily
to the first regions 22 corresponding to the positions of the
microchambers 14.
[0054] Here, as methods for forming the first regions 22 and the
second region 23, the following examples (1) to (4) are
conceivable. Note that these structures may be combined
appropriately
[0055] (1) Hydrophilicity is given to the first regions 22 to
increase absorption of protein, making the adherence to cells in
the first regions 22 relatively high. For example, the first
regions 22 with hydrophilicity can be formed by patterning by
irradiating the culture plane of the culture container 13 with
plasma or ultraviolet rays.
[0056] (2) A polymeric film having an electric charge (for example,
a coating of polylysine) is formed on the first regions 22 to
increase absorption of protein, making the adherence to cells in
the first regions 22 relatively high.
[0057] (3) A film which inhibits absorption of protein is formed on
the second region 23, thereby making the adherence to cells in the
second region 23 relatively low. Examples of material of the film
include polyethylene glycol (PEG), 2-methacryloyloxyethyl
phosphorylcholine (MPC), and the like.
[0058] (4) Minute projections may be formed in the second region 23
by surface treatment to decrease the area for cells to adhere,
making the adherence to cells in the second region 23 relatively
low.
[0059] Note that procedures of the method for measuring activity in
another embodiment are mostly common to the first embodiment except
that the microchamber sheet 21 is disposed in the culture container
13 and that the environmental factors are measured in each of the
individual microchambers 14, and thus the description thereof is
omitted. In addition, when alignment marks are provided in advance
on the microchamber sheet 21 or the culture container 13,
positioning becomes easy when the microchamber sheet 21 is disposed
in the culture container 13.
[0060] According to another embodiment, in addition to the effects
of one embodiment, measurement of activity of cells can be
performed at once in arrays. Thus, throughput for evaluating
activity of plural cells can be increased significantly. Further,
in another embodiment, by having the arrays, it is unnecessary to
align the individual microchambers 14 separately. Thus, complexity
of measurement work decreases, and working efficiency improves
further.
[0061] <Structure Example of a Device for Measuring
Activity>
[0062] FIG. 8 is a block diagram illustrating a structure example
of a device for measuring activity for carrying out the method for
measuring activity of the above-described one embodiment or another
embodiment.
[0063] The device for measuring activity has a
temperature-controlled room 31, a robot arm 32, an optical
observation unit 33, a monitor 34, a memory unit 35, a controlling
unit 36, and an operation unit 37 accepting various operations from
the user. Here, the robot arm 32, the optical observation unit 33,
the monitor 34, the memory unit 35, and the operation unit 37 are
connected to the controlling unit 36.
[0064] The temperature-controlled room 31 accommodates the culture
container 13 in which cells 12 to be measured are incubated. Inside
this temperature-controlled room 31, an environment suitable to
incubation of cells (for example, an atmosphere at a temperature of
37.degree. C. and at a humidity of 90%) is maintained, and high
cleanliness is maintained for preventing contamination.
[0065] The robot arm 32 holds the microchamber 14 (or the
microchamber sheet 21) on its front end, and moves the microchamber
14 three-dimensionally. In response to instructions from the
controlling unit 36, the robot arm 32 performs operations to place
the microchamber 14 to cover a cell 12 at a predetermined position
in the culture container 13, and to remove the microchamber 14
after measurement is finished.
[0066] The optical observation unit 33 has an illumination device
lighting cultured cells, a microscopic optical system for observing
the cultured cells, and an imaging device imaging an object in the
culture container 13 via the microscopic optical system. This
optical observation unit 33 is used for obtaining position
information when the robot arm 32 positions the microchamber 14 in
the culture container 13, and for obtaining image information
indicating an optical change occurring in the measurement space in
the microchamber 14. Here, the image taken in the optical
observation unit 33 can be displayed on the monitor 34 under
control of the controlling unit 36.
[0067] The memory unit 35 is formed of a non-volatile storage
medium such as a hard disk or a flash memory. In this memory unit
35, measurement information of the environmental factors and image
information generated by the optical observation unit 33 during
measurement of the environmental factors are stored. The image
information and the measurement information of environmental
factors are stored in the memory unit 35 in a state of being
correlated with identification information of the cells to be
measured and measurement date and time information. In the memory
unit 35, a program to be executed by the controlling unit 36 is
also stored.
[0068] The controlling unit 36 is a processor controlling
operations of the respective parts of the device for measuring
activity in a centralized manner. For example, the controlling unit
36 controls the robot arm 32 to move the microchamber 14. Further,
the controlling unit 36 performs measurement of the environmental
factors in the microchamber 14 with the imaging device of the
optical observation unit 33 and the environmental factor sensor 15
in the microchamber 14.
[0069] Hereinafter, an operation example of the device for
measuring activity will be described with reference to the
flowchart of FIG. 9.
[0070] Step S101: the controlling unit 36 activates the optical
observation unit 33 to obtain an observation image capturing the
state in the culture container 13. Thus, the controlling unit 36
obtains the position information for positioning the microchamber
14. For example, the controlling unit 36 correlates a reference
point of an image (for example, the center of an image) with a
position in the culture container 13 in advance. Then the
controlling unit 36 can geometrically obtain actual coordinates of
a cell in the culture container 13 from the positional relationship
between the cell in the image and the reference point, considering
magnifying power, lens position, and so on in the optical
observation unit 33.
[0071] Thereafter, the controlling unit 36 displays the
aforementioned observation image on the monitor 34. Accordingly,
the user can specify the cell 12 to be measured with the operation
unit 37 based on the observation image.
[0072] Step S102: upon reception of the specification of the cell
12 to be measured from the user, the controlling unit 36 drives the
robot arm 32 based on the aforementioned position information
(S101) to dispose the microchamber 14 from an upper side of the
cell 12 to be measured. Thus, the measurement space is formed by
the microchamber 14 surrounding the cell 12 to be measured.
[0073] Step S103: the controlling unit 36 carries out measurement
of the environmental factors in the measurement space by taking an
image with the optical observation unit 33 and by outputs of the
environmental factor sensor 15. After statistically analyzing
measurement results of the environmental factors as necessary, the
controlling unit 36 records the measurement results in the memory
unit 35 and displays the measurement results on the monitor 34.
[0074] Step S104: after the measurement is completed, the
controlling unit 36 drives the robot arm 32 to remove the
microchamber 14 from the culture container 13, and recovers the
incubating environment in the culture container 13.
[0075] Thus, the description of the flowchart of FIG. 9 is
completed. Using the above-described device for measuring activity,
it is possible to carry out the method for measuring activity of
the above-described embodiment efficiently.
Example
[0076] As an example, a calibration curve of an oxygen sensor was
created, so as to demonstrate that measurement of a dissolved
oxygen concentration in the microchamber (reactor) is possible.
[0077] FIG. 10 is a schematic diagram illustrating the structure of
a measurement system in the example. In the example, a sheet of
polydimethylsiloxane (PDMS) containing a ruthenium (Ru) complex is
disposed on a glass substrate, and this glass substrate on which
the aforementioned sheet is disposed and a silicon (Si) wafer in
which a hole having a diameter of 500 .mu.m to 800 .mu.m is bored
are bonded together to form a reactor. Further, an upper side of
the Si wafer is enclosed with a glass substrate. Here, the
thickness of the entire reactor is 500 .mu.m.
[0078] Solutions having different dissolved oxygen concentrations
were enclosed in the aforementioned reactor, and fluorescence
intensity in each of them was measured with a microscope.
[0079] FIG. 11 is a graph illustrating a relation between
fluorescence intensity and a dissolved oxygen concentration in the
example. From FIG. 11, it is recognized that the fluorescence
intensity decreases as the dissolved oxygen concentration in the
reactor increases.
[0080] Next, the calibration curve of the oxygen sensor is created
based on experimental data from the example. A parameter of the
calibration curve is represented with the following equation
(1).
I.sub.0/I=1+K.sub.SV[O.sub.2] (1)
[0081] Here, "I.sub.0" denotes the fluorescence intensity when the
dissolved oxygen concentration is 0 mg/L. "I" denotes the
fluorescence intensity with respect to each dissolved oxygen
concentration. "K.sub.SV" denotes a value indicating an inclination
of the calibration curve. "O.sub.2" denotes the dissolved oxygen
concentration.
[0082] In the example, since the fluorescence intensity when the
dissolved oxygen concentration is 0 mg/L cannot be obtained by
experiment, the inverse number of the fluorescence intensity is
approximated with a straight line (see FIG. 12), and I'' obtained
from an intercept y of an approximation curve is assumed as the
I.sub.0.
I''=1/(2.159.times.10.sup.-5)=46317.74 (2)
[0083] Using the aforementioned I'', the calibration curve was
created with a vertical axis being I''/I and a horizontal axis
being the dissolved oxygen concentration (see FIG. 13). At this
time, K.sub.SV=0.19976.
[0084] <Supplemental Items of the Embodiments>
[0085] (1) In the above-described embodiments, when a light curing
resin is used for the microchamber 14, an arbitrary cell can be
enclosed in the microchamber 14 by irradiating the microchamber 14
with laser. With this structure, it becomes possible to selectively
separate and extract a desired cell based on measurement results of
environmental factors in the measurement space.
[0086] (2) In the example of FIG. 4, an opening part covered with a
hydrophobic semi-permeable film 17 may be formed on a side face of
the microchamber 14.
[0087] (3) In the above-described embodiments, when fluorescence in
the microchamber 14 is observed, a reflective film reflecting the
wavelength of fluorescence may be formed on the inside of a side
wall of the microchamber 14. With this structure, it becomes
possible to detect a fluorescence signal in the microchamber 14
with higher sensitivity.
[0088] (4) In addition, it is preferred that the instruments of the
above-described embodiments, such as the microchamber 14, the
microchamber sheet 21, the culture container 13, and so on, be
designed as a disposal item.
[0089] The many features and advantages of the embodiments are
apparent from the detailed specification and, thus, it is intended
by the appended claims to cover all such features and advantages of
the embodiments that fall within the true spirit and scope thereof.
Further, since numerous modifications and changes will readily
occur to those skilled in the art, it is not desired to limit the
inventive embodiments to the exact construction and operation
illustrated and described, and accordingly all suitable
modifications and equivalents may be resorted to, falling within
the scope thereof.
* * * * *