U.S. patent application number 15/478041 was filed with the patent office on 2017-07-20 for data analysis of cells undergoing excitation contraction coupling as detected on real-time cell analysis (rtca) instruments.
The applicant listed for this patent is ACEA Biosciences, Inc.. Invention is credited to Yama A. Abassi, Nan Li, Xiaobo Wang, Biao Xi, Xiao Xu, Wen Fu Zhang.
Application Number | 20170205391 15/478041 |
Document ID | / |
Family ID | 44992027 |
Filed Date | 2017-07-20 |
United States Patent
Application |
20170205391 |
Kind Code |
A1 |
Li; Nan ; et al. |
July 20, 2017 |
DATA ANALYSIS OF CELLS UNDERGOING EXCITATION CONTRACTION COUPLING
AS DETECTED ON REAL-TIME CELL ANALYSIS (RTCA) INSTRUMENTS
Abstract
A method of determining a beating parameter of cells that
undergo excitation contraction coupling, the method including
providing a cell analysis device having a substrate and a sensor
that measures cell adhesion or attachment to the substrate in
millisecond time resolution; adding excitable cells capable of
undergoing excitation contraction coupling to the substrate;
monitoring cell adhesion or attachment of the excitable cells to
the substrate in millisecond time resolution; and calculating one
or more beating parameters from the monitored adhesion.
Inventors: |
Li; Nan; (San Diego, CA)
; Wang; Xiaobo; (San Diego, CA) ; Abassi; Yama
A.; (San Diego, CA) ; Xi; Biao; (San Diego,
CA) ; Zhang; Wen Fu; (San Diego, CA) ; Xu;
Xiao; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ACEA Biosciences, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
44992027 |
Appl. No.: |
15/478041 |
Filed: |
April 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13109809 |
May 17, 2011 |
9612234 |
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15478041 |
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12435569 |
May 5, 2009 |
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13109809 |
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61191684 |
Sep 11, 2008 |
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61126533 |
May 5, 2008 |
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61345867 |
May 18, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/48728 20130101;
G01N 33/5008 20130101; G01N 2800/326 20130101; G01N 33/4836
20130101; G01N 33/5014 20130101; G01N 33/5073 20130101; G01N
2800/52 20130101; G01N 33/5061 20130101 |
International
Class: |
G01N 33/487 20060101
G01N033/487; G01N 33/50 20060101 G01N033/50 |
Claims
1. A method of determining a beating parameter of cells that
undergo excitation contraction coupling, the method comprising: a)
providing a cell analysis device comprising a substrate and a
sensor that measures cell adhesion or attachment to the substrate
in millisecond time resolution; b) adding excitable cells capable
of undergoing excitation contraction coupling to the substrate; c)
monitoring cell adhesion or attachment of the excitable cells to
the substrate in millisecond time resolution; and d) calculating
one or more beating parameters from the monitored adhesion.
2. The method according to claim 1, wherein the cell analysis
device is an impedance monitoring device that measures
cell-substrate impedance.
3. The method according to claim 1, wherein the cells are
cardiomyocytes.
4. The method according to claim 1, wherein the cells are stem cell
derived cardiomyocytes.
5. The method according to claim 1, wherein the cells are induced
pluripotent stem cell (iPS cell) derived cardiomyocytes.
6. The method according to claim 1, wherein the millisecond
resolution is characterized as consecutive impedance measurements
less than 40 milliseconds apart.
7. The method according to claim 6, wherein the millisecond
resolution is characterized as consecutive impedance measurements
less than 20 milliseconds apart.
8. The method according to claim 1, wherein the step of calculating
one or more parameters comprises forming a beating curve from the
monitored cell adhesion or attachment and deriving the one or more
beating parameters from the beating curve.
9. The method according to claim 1, wherein the one or more beating
parameters are selected from the group consisting of beating rate,
beating amplitude, rising time, falling time, beating period,
IBD10, IBD50, IBD90, rising slope and falling slope.
10. The method according to claim 1, wherein the one or more
beating parameters are selected from the group consisting of
normalized beating rate, normalized beating amplitude, beating
pattern similarity and beating rhythm irregularity.
11. The method according to claim 1, wherein the one or more
beating parameters comprises 5 beating parameters.
12. The method according to claim 1, wherein the one or more
beating parameters are calculated while the device continues to
monitor the cell adhesion or attachment.
13. The method according to claim 1, further comprising adding a
test compound to the cells, wherein the step of monitoring cell
adhesion or attachment of the excitable cells is performed after
adding the test compound.
14. The method according to claim 13, wherein the step of
monitoring cell adhesion or attachment of the excitable cells is
performed before and after adding the test compound.
15. A method of determining a dose response for a test compound on
a cell population, the method comprising: a) determining a beating
parameter for different doses of a test compound according to the
method of claim 1, wherein the test compound is added at different
doses to different populations of cells; and b) plotting the
beating parameters for each dose to form a dose response curve.
16. The method according to claim 15, wherein the beating parameter
for each dose is from a same time point.
17. The method according to claim 15, further comprising
determining a IC50 or EC50 value from the dose response curve.
18. A method for identifying a compound having a potentially
cardiotoxic effect, comprising: a) providing a test compound
suspected of having a cardiotoxic effect; b) performing the method
according to claim 1 to obtain a beating parameter for the test
compound, wherein the cells are cardiomyocytes and wherein the test
compound is added to the cardiomyocytes; and c) comparing the
beating parameter for the test compound to a control beating
parameter to identify whether there is a difference between beating
parameters and if so, concluding the test compound has the
potentially cardiotoxic effect.
19. The method according to claim 18, wherein the beating parameter
for the test compound comprises 2 beating parameters, which are
compared to 2 control beating parameters, further wherein the test
compound is concluded to have a cardiotoxic effect if at least one
of the compared parameters is different.
20. The method according to claim 18, wherein the compound is
suspected of being a pro-arrhythmic drug that may induce
arrhythmia.
21. The method according to claim 18, wherein the step of
monitoring the beating of cells is also performed before adding the
test compound.
22. The method according to claim 18, wherein the test compound is
added at different doses to the cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. Ser. No. 13/109,809, filed
May 17, 2011, which is a continuation in part of U.S. patent
application Ser. No. 12/435,569, filed on May 5, 2009, which claims
benefit of priority to U.S. provisional patent application Ser. No.
61/191,684, filed on Sep. 11, 2008 and U.S. provisional patent
application Ser. No. 61/126,533, filed on May 5, 2008, the contents
of each are herein incorporated by reference in their entirety.
[0002] U.S. Ser. No. 13/109,809 also claims benefit of priority to
U.S. provisional patent application Ser. No. 61/345,867 filed on
May 18, 2010; the contents of which are herein incorporated by
reference in their entirety.
TECHNICAL FIELD
[0003] The invention relates to cell based assays that monitor
excitation contraction coupling of cells and more specifically to
methods for determining cell beating parameters by monitoring cell
adhesion or attachment of cells that undergo excitation contraction
coupling.
BACKGROUND OF THE INVENTION
[0004] Cardiac safety pharmacology is the study of the potential
undesirable pharmacodynamic effects of a substance on heart
function in relation to exposure to the substance in the
therapeutic range and above. Cardiac safety is a major concern in
current drug development. Since 1981, at least 10 blockbuster drugs
have been withdrawn from the market due to cardiac liability,
defined as potentially undesirable effects on heart function.
Furthermore, cardiac safety is a major reason for late stage
attrition of drug candidates during development.
[0005] There are three non-mutually exclusive ways that non-cardiac
drugs may lead to cardiac liability. Directly, cardiotoxic drugs
are drugs which cause damage via necrosis or apoptosis, such as
anthracyclines. Pro-arrhythmic drugs are drugs which induce
arrhythmia. Indirectly, cardiotoxic drugs are drugs which
indirectly affect cardiac function, such as by causing narrowing of
the arteries.
[0006] Directly, cardiotoxic drugs directly affect the viability of
cardiomyocytes and therefore heart function. A prominent class of
drugs in this category is chemotherapeutic drugs, such as
anthracyclines. Mortality due to cardiac disease is thought to be
8-fold higher for survivors of childhood cancers who have received
chemotherapy. These drugs are thought to disrupt iron metabolism,
generating harmful oxygen radical species which ultimately cause
mitochondrial damage and apoptosis.
[0007] Pro-arrhythmic drugs induce arrhythmia. Normal synchronized
contractile activity of cardiomyocytes is the result of
orchestrated ion currents passing across the cell membrane via
ion-specific channels and coupling with the specialized
cytoskeleton. Disturbances in the ionic movement of interference
with ion channel activities may lead to arrhythmia. It is believed
that one of the primary targets of pro-arrhythmic drugs is the ERG
channel, which is responsible for delayed repolarization of
cardiomyocytes. ERG channel blockage may lead to QT elongation and
this may cause a fatal form of ventricular arrhythmia called
Torsades de Pointes (TdP). Between 1990 and 2006, 10 blockbuster
drugs have been withdrawn from the market due to induction of TdP.
The drugs that have been associated with cardiac arrhythmia and
removed from the market are prenylamine, terodiline, sparfloxacin,
sertindole, terfenadine, astemizole, grepafloxacin, cisapride,
droperidol, and levacetylmethadol.
[0008] Excitation-contraction coupling (ECC) is a term used to
describe the physiological process of converting an electrical
stimulus to a mechanical response. The process is fundamental to
muscle physiology, wherein the electrical stimulus may be an action
potential and the mechanical response is in the form of
contraction. Although ECC has been known generally over half a
century, it is still an active area of biomedical research.
[0009] Cardiomyocytes are specialized muscle cells of the
myocardium that are capable of excitation-contraction coupling.
Cardiomyocytes are commonly used in biomedical research to assess
the cardiotoxicity of potential drugs or treatments. Two
conventional approaches to assess cardiotoxicity are primarily
used. A first approach involves isolation of cardiomyocytes
directly from a mammalian species such as rats and dogs followed by
electrophysiological studies on the isolated cardiomyocytes.
However, this approach suffers from being extremely
labor-intensive, time consuming and costly and at the same time not
very amenable to the high throughput demands of pharmaceutical
industry. An alternative approach utilizes cell-based assay models,
which heterologously express specific ion channels such as hERG
channels or voltage-gated calcium channels. These cardiac ion
channels have been envisioned as possible molecular targets through
which drugs could induce cytotoxicity. These cell-based systems
allow assessment of drug-channel interaction by monitoring the
effect of the drug on currents produced by different channels in
cultured cells using a technique known as "patch clamping." Patch
clamping isolates regions of the cell membrane containing channel
proteins and measures changes in electrical potential difference.
However, use of this method in high throughput requires automation
of patch clamping in an array format with reliable giga seal, which
even though is becoming increasing available, is not yet
widespread. In addition, cardiac toxicity may occur by other
mechanisms that could be possibly missed by this type of targeted
approach. An alternative to in vitro ion-channel recording assays
as well as the labor-intensive isolation of primary tissue is the
differentiation of embryonic stem (ES) cells into cardiomyocytes.
The utility of ES cells as a treatment for various chronic diseases
has received much attention in recent years. Mammalian ES cells are
self renewing cells derived from the inner cell mass of a
blastocyst stage embryo which can be differentiated into multiple
different cell types. It has been demonstrated that the mouse ES
cells as well as human ES cells can be differentiated into
cardiomyocytes which retain the ability to beat in culture.
Differentiation of ES cells first involves an intermediate in vitro
developmental stage in which ES cells form compact cell structures
known as embryoid bodies. These embryoid bodies can induce the
developmental program of ES cell differentiation into multiple cell
types including cardiomyocytes, which are distinguished in culture
by their ability to undergo spontaneous beating. These ES derived
in vitro differentiated cardiomyocytes recapitulates the normal
development of cardiomyocytes as evidenced by the stage-specific
expression of cardiomyocyte specific genes. All the known
transcription factors, ion channels and structural proteins that
are part of normal heart development and function in vivo are also
expressed in ES-derived cardiomyocytes.
[0010] Even though high throughput to medium throughput systems
have been developed for functional characterization of cell lines
heterologously expressing the gene for specific ion channels, high
throughput techniques for functional characterization of more
complex systems such as cardiomyocytes have been limited.
Technologies designed to assess cardiomyocyte behaviour and
function and the effect of drugs and other manipulations in vitro
can be divided into two different approaches. One approach involves
long term assessment of cardiomyocyte viability for example in
response to certain compounds. Such assays are typically end point
assays designed to measure a cellular component such as ATP which
correlates with the degree of viability of the cells. The other
approach involves studying short term effect of drugs and compounds
on beating function of cardiomyocytes. High throughput techniques
for short term functional characterization of ion channels and
other targets in cardiomyocytes has been rather challenging and
limited. The available systems typically only monitor a single
cardiomyocyte or a small number of cardiomyocytes at a time with
very limited throughput.
SUMMARY OF THE INVENTION
[0011] The invention addresses the need for further study of
cardiomyocytes and their response to therapeutic agents by
establishing a number beating parameters which may be used to
assess the heath, function and response of the cardiomyocytes to
potential treatment of drugs or chemical compounds. The above is
accomplished by providing a method of determining one or more
beating parameters for use in cardiomyocyte beating analysis. The
method includes providing a cell analysis device including wells,
each well including a sensor capable of monitoring beating of
cardiomyoctes in millisecond time resolution; adding cardiomyocytes
to the wells; monitoring the beating of the cardiomyocytes in
millisecond time resolution to obtain a plurality of beating
measurements; and calculating one or more beating parameters from
the plurality of beating measurements.
[0012] The cell analysis device should be able to measure
cardiomyocytes beating in millisecond time resolution. In some
embodiments, the cell analysis device is an impedance monitoring
device, such as an impedance monitoring device having a sensor
formed from two electrode structures, each having substantially the
same surface area. In other embodiments, the sensor is an optical
sensor. Examples of suitable optical sensors are those that can be
used to detect or measure changes in cell morphology, cell adhesion
and cell number. Among these include a resonant waveguide or a
resonant waveguide grating sensor.
[0013] In some embodiments, beating measurements are plotted over
time to form, at least in part a beating curve. Beating parameters
can then be derived from the curve or corresponding data. Among the
beating parameters of particular interest, include beating rate,
beating amplitude, rising time, falling time, beating period,
IBD50, IBD10, IBD90, rising slope, falling slope, normalized
beating rate, normalized beating amplitude, beating pattern
similarity and beating rhythm irregularity.
[0014] Test compounds may be added to identify changes in beating
parameters and thus the effect on cardiomyocytes. Accordingly, in
some embodiments a test compound is added to the wells at a number
of different concentrations and a dose response curve (DRC) is
constructed. In further embodiments, the methods include
determining an IC50 or EC50 value from the dose response curve. In
other embodiments a test compound is added to the wells to assess a
potential difference in beating parameters, which may be indicative
of cardiotoxicity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-B depict schematic drawings of one design of a
cell-substrate impedance measurement device of the present
invention. FIG. 1A shows a nonconductive substrate 101 with 16
electrode arrays or sensors fabricated on the substrate. Each
electrode array 102 includes two electrode structures. Each
electrode structure comprises multiple electrode elements. Each
electrode array connects to two electrical traces 103, with each of
the two traces connected one of the two electrode structures. These
electrical connection traces 103 from the electrode array 102 are
connected to the connection pads 104 at the edges of the substrate
101. As shown in FIG. 1A, each of the four electrode arrays in each
of four quarters on the substrate 101 have one of their electrical
connection traces 103 connected to a common connection pad 104.
Thus, for the depicted device there are four common connection pads
104, one for each quarter of the device. In addition, each
electrode array has a separate electrical connection trace 103,
connecting to an independent connection pad 104. Thus, there are
total 20 connection pads 104 at the edges of the substrate (101).
In FIG. 1B a single exemplary electrode array or sensor is shown.
The electrode array has two electrode structures, where each
electrode structure comprises multiple electrode elements 105 shown
here having a circle-on-line geometry. In this electrode array
structure, electrode elements 105 of one electrode structure of the
array alternate with electrode elements 105 of the other electrode
structure of the array. Each of the electrode structures is
independently connected to its electrode bus 106, in this case, by
means of direct connection of the electrode elements 105 to the
electrode bus 106. Each electrode bus 106 forms an arc around the
perimeter of the array, where the two buses of the array do not
abut or overlap. The electrically conductive connection traces 103
in FIG. 1A connect each bus with a connection pad 104 in FIG. 1A on
the edge of the substrate 101 in FIG. 1A.
[0016] FIG. 2 depicts graphs demonstrating combined long term and
short term measurement of cardiomyocytes beating over at least 7
days including measurement in millisecond time resolution with
corresponding cell index values calculated from impedance
measurements over beating periods at days 3, 5 and 7.
[0017] FIG. 3 depicts graphs showing change in cell index in
millisecond time resolution before and after adding 190 nM of
compound E4031, which is has pro-arrhythmic effects.
[0018] FIG. 4 depicts a series of graphs showing the effect on cell
index of a cardiomyocyte cell population in millisecond time
resolution before and after adding petamidine, which prevents ERG
transport to the membrane and thus a delayed adverse effect over 6
hours.
[0019] FIG. 5 depicts a series of graphs showing change in cell
index in millisecond time resolution of iPS cell-derived
cardiomyocytes and monitoring of the cardiomyocytes over 45
days.
[0020] FIG. 6 depicts millisecond time resolution charting of cell
index of cardiomyocytes tested with increasing concentration of
pro-arrhythmic drugs withdrawn from the US market, namely
Droperidol, Astemizole, Cisapride, Sertindole and Terodiline.
[0021] FIG. 7 depicts a graphical representation of multiple
cardiomyocyte beating periods and graphical illustration of
features used in beating parameters including positive peaks P1,
P2, P3 . . . Pm; negative peaks -P1, -P2, -P3, . . . -Pm; and
beating amplitude Amp-1, Amp-2, Amp-3 . . . Amp-m.
[0022] FIG. 8 depicts a graphical representation of a portion of a
beating period to demonstrate a rise and fall of beating amplitude
including time points for 20% amplitude, 50% amplitude and 80%
amplitude.
[0023] FIGS. 9A-D depicts graphs and an accompanying table
resulting from dynamic monitoring and characterization of mouse
embryonic stem cell derived cardiomyocytes (mESCC) beating using
impedance-based detection. FIG. 9A shows long term impedance
monitoring of cells with arrows depicting time points for short
term millisecond recording. FIG. 9B shows beating activity and
profile of mESCC at time points identified in FIG. 9A after cell
seeding. The beating rate (1/min), amplitude (delta CI), beat
duration (IBD50; ms), Time to Max (ms) and Decay Time (ms) were
quantified using the RTCA Cardio software and as described in the
Examples section. The data represents the average of 8 wells
-/+Standard Deviation. A total duration of 5 sec recording time is
displayed. FIG. 9C demonstrates blebbistatin, an inhibitor of
myosin heavy chain ATPase activity, inhibits beating activity of
mESCC, which is restored by washing out the compound and replacing
by normal growth media. FIG. 9D demonstrates that Blebibistain
treatment of mESCC has no effect on field potential recording as
measured on Multi-Electrode Arrays.
[0024] FIGS. 10A-C depict graphs showing pharmacological assessment
of ion channel modulators measured using the impedance-based system
on mouse embryonic stem cell derived cardiomyocytes (mESCC). Cells
were seeded in the wells of the E-PLATE (ACEA Biosciences Inc., San
Diego, Calif.), monitored for 3 days using the RTCA Cardio system
and treated with the indicated concentrations of each compound. The
beating activity was recorded by the RTCA Cardio system as
described in the Examples section. For each compound at the
indicated time points 5 sec of beating activity is displayed with
the exception of ERG which is total of 14 seconds of beating
activity. The beating rate for each interval of beating activity is
displayed as beats/min-/+SD; the data shown is one representative
recording from a total of at least 3 separate experiments. FIG. 10A
shows results from treatment with Isrdapine, an L-Type
voltage-gated calcium channel inhibitor, FIG. 10B shows results
from treatment with (S)-(-)Bay K 8644, an agonist of L-Type
voltage-gated calcium channels, FIG. 10C shows results from
treatment with chromanol, inhibitor of the slow delayed rectifier
K.sup.+ current.
[0025] FIGS. 11A-C depict graphs showing pharmacological assessment
of ERG channels, sodium channels and ionotropic agents. FIG. 11A
shows results from treatment with E4031, an inhibitor of ERG type
K.sup.+ channel, FIG. 11B shows results from treatment with
Tetrodotoxin (TTX), inhibitor of voltage-gated Na.sup.+ channel,
and FIG. 11C shows results from treatment with isoproterenol, an
ionotropic agent and agonist of the .beta.-adrenergic receptor.
[0026] FIG. 12 depicts a table summarizing beating parameters
including normalized beating rate, normalized amplitude and beating
rate irregularity of the compounds tested in FIGS. 10A-C and FIGS.
11A-C.
[0027] FIGS. 13A and B depict graphs showing mechanism-based
cardiotoxicity profiling of cardiomyocytes using impedance-based
devices. FIG. 13A shows screening, in a dose-response manner in
mESCC, 4 drugs which have been withdrawn from the market due to
increased incidence of TdP arrhythmia. For each compound a total of
5 sec of beating activity is displayed. For Astemizole, cisapride,
droperide and sertindole the dose-response profiles are shown at 30
min, 15 min, 180 min and 165 min after compound addition,
respectively. The bottom row shows the dose-response for each of
the compounds at the indicated time points based on calculation of
beat duration parameter as described in the Examples section. In
FIG. 13B plateau oscillation profiles are induced by all four
compounds tested in FIG. 13A as well as E-4031, indicating a common
underlying mechanism; at total of 9 sec of beating profile
recording is displayed for each of the compounds.
[0028] FIGS. 14A-C depict graphs and results from treatment with
doxorubicin and pentamidine. FIG. 14A shows mESCC treated with
increasing concentrations of doxorubicin. Dose-dependent
impedance-based cellular profiles were monitored for up to 24 hours
after compound treatment. In FIG. 14B a total of 5 sec of recording
is shown for each dose at the given time point. The beating
activity is quantified in terms of beating rate and displayed in
each box. In FIG. 14C mESCC were seeded in the wells of E-PLATE
(ACEA Biosciences, San Diego, Calif.) and on day 3 treated with 20
.mu.M Pentamidine. The beating activity was monitored at the
indicated time windows after compound treatment and quantified
based on beat duration as mentioned in the Examples section.
[0029] FIG. 15 depicts a table summarizing the screening results of
50 compounds with potential cardiotoxic liability screened at 3
final doses (10 .mu.M, 1 .mu.M and 0.1 .mu.M) using the RTCA Cardio
system together with mESCC. For each compound both the lowest
observed concentration (LOC) which resulted in an effect on mESCC
and the resulting beating profile was included.
DETAILED DESCRIPTION
[0030] The present invention addresses the need to provide methods
to further improve monitoring of excitation-contraction coupling in
cardiomyocytes and excitable cells, such as for cardiac safety
assessment and for the study of cardimyocyte function and
differentiation. Specifically, embodiments of the invention
describe label-free methods for monitoring cardiomyocytes in vitro
and methods for effective comparison by establishing a plurality of
beating parameters. The methods are capable of continuously
monitoring excitation-contraction coupling and determining the
beating parameters in a relatively high-throughput manner, which
offers a unique approach to assessing the safety of potentially
cardiotoxic treatments, which may not be readily apparent. The
systems and methods can be used to assess both short term and long
term effects of substances on cardiomyocyte beating, viability and
morphology.
Monitoring Beating of Cardiomyocytes
[0031] While the invention relates primarily to methods for
determining beating parameters for beating analysis and comparison,
it is important to obtain reliable beating measurements in
millisecond time resolution. Millisecond time resolution preferably
refers to the measurement or acquisition of data from at least two
consecutive time points within 100 milliseconds of one another.
More preferably, the at least two consecutive beating measurements
are performed in less than 40 milliseconds. Still more preferably,
the at least two consecutive measurements are performed in less
than 20 milliseconds. In still further embodiments the at least two
consecutive beating measurements are performed in less 10
milliseconds.
[0032] In one approach a device for monitoring the beating of cells
may be an optical-based system, which uses an optical sensor.
Optical sensors typically measure an optical property of the cell,
which may relate cell morphology, cell adhesion degree and/or cell
number. Among the optical sensors that may be used include resonant
waveguide sensors or resonant wave guide grating sensors. For
example, the resonant wave guide grating sensor utilizes the
resonant coupling of light into a waveguide through a diffraction
grating. A polarized light, covering a range of incident
wavelengths, is directly used to illuminate the waveguide; light at
specific wavelengths is coupled into and propagates along the
waveguide. The resonance wavelength at which a maximum in coupling
efficiency is achieved is a function of the local refractive index
at or near the sensor surface. When the cardiomyocytes are cultured
and attached to the surfaces of the resonant wave guide sensor, the
local refractive index on the sensor/cell interface would be
affected by changes in cell morphology, cell adhesion and other
cellular properties. For example, a relocation or re-arrangement of
certain cellular contents such as the change in cell adhesion
degree, or membrane refilling, recruitment of intracellular
proteins to activated receptors at the cell surface, or receptor
endocytosis, the change in cell morphology may all result in the
change in local refractive index, leading to a detectable change or
shift in the maximum in coupling efficiency wavelength. The effect
of the presence of the cells on a resonant wave guide grating
sensor has been explored based on the dynamic mass redistribution
model. The details of such optical sensors and other optical
sensors for cell-based assays have been described in detail in
"Label-free cell-based assays with optical biosensors in drug
discovery", by Ye Fang, in Assay and Drug Development Technologies,
Vol 4, pp 583-595, 2006. An optical-sensor based system may include
a device comprising optical sensors in wells suitable for cell
culture, an optical signal measurement/detection system such as
optical CCD camera, an optical-signal processing algorithm to
process optical signals in short-time resolution, such as
millisecond resolution, to provide a cardiomyocyte-beating
dependent curve, and to quantify cell beating and to derive
cell-beating parameters (such as calculating average rate of beats
per unit time, average amplitude intensity in a unit time as well
as the average length of time between the beats) based on cell
morphology images.
[0033] In the preferred approach the system is an impedance-based
cell monitoring system. Most preferably, the system provides a
device for monitoring cell-substrate impedance, an impedance
analyzer capable of impedance measurements at millisecond time
resolution, electronic circuitry that can engage the device and
selectively connect two or more sensors or electrode arrays of the
device to the impedance analyzer and a software program that
controls the electronic circuitry and records and analyzes data
obtained from the impedance analyzer. By providing the impedance
based system with millisecond time resolution, beating measurements
from excitation-contraction coupling of cells can be efficiently
monitored in millisecond time resolution. Thus the methods provided
herein may be combined with impedance-based systems to identify and
evaluate changes in excitation-contraction events and shifts in
beating parameters discussed below, which may be used for high
throughput analysis of potential therapeutics. The skilled artisan
will appreciate that millisecond time resolution measurements may
be coupled with longer term impedance monitoring, such as longer
than seconds, hours or days. In some embodiments, long term
impedance monitoring is performed with intermittent periods of
impedance monitoring at millisecond time resolution. Suitable
impedance monitoring systems are described in detail in U.S. patent
application Ser. No. 12/435,569, which is herein incorporated by
reference in its entirety. A preferred electrode array or
configuration is also provided in FIGS. 1A and B.
[0034] The preferred device for monitoring substrate impedance
includes a nonconductive substrate having one or more individually
addressable electrode arrays or sensors fabricated thereon and one
or more wells. A surface of the substrate may be suitable for cell
attachment, wherein the cell attachment results in a detectable
change in impedance between electrodes within the array.
Preferably, the nonconducting substrate is planar, and is flat or
approximately flat. The substrates may be constructed from a
variety of nonconductive materials known in the present art,
including, but not limited to, silicon dioxide on silicon,
silicon-on-insulator (SOI) wafer, glass (e.g., quartz glass, lead
glass or borosilicate glass), sapphire, ceramics, polymer, fiber
glass, plastics, e.g., polyimide (e.g. Kapton, polyimide film
supplied by DuPont), polystyrene, polycarbonate, polyvinyl
chloride, polyester, polypropylene and urea resin. Preferably, the
substrate is biocompatible with excitable cells such as
cardiomyocytes; however, materials that are not biocompatible can
be made biocompatible by coating with a suitable material, such as
a biocompatible polymer or coating. Further, attachment or growth
along the substrate or electrodes may be enhanced by pre-coating
the substrate with a protein or compound that facilitates
attachment or growth. Such compounds may be chosen according to
techniques known in the cellular biology arts; however, in some
embodiments fibronectin is effective. Alternatively, the substrate
may be chemically modified to display reactive groups that enhance
cell attachment, particularly ES cells or cardiomyocytes.
[0035] Preferably, each sensor or electrode array includes two or
more electrode structures that are constructed to have dimensions
and spacing such that they can, when connected to a signal source,
operate as a unit to generate an electrical field in the region of
spaces around the electrode structures. Preferably the electric
field is substantially uniform across the array. An electrode
structure refers to a single electrode, particularly one with a
complex structure. Specifically, an electrode array includes two
electrode structures, each of which includes multiple electrode
elements, or substructures, which branch from the electrode
structure. In preferred embodiments, the electrode structures of
each of the two or more electrode arrays of a device have
substantially the same surface area.
[0036] Each of the two electrode structures of an electrode array
is connected to a separate connection pad that is preferably
located at the edge of the substrate. Specifically, for each of the
two or more electrode arrays of the device, preferably the first of
the two electrode structures is connected to one of the two or more
connection pads, and the second of the two electrode structures is
connected to another of the two or more connection pads.
Preferably, each array of a device is individually addressed,
meaning that the electrical traces and connection pads of the
arrays are configured such that an array can be connected to an
impedance analyzer in such a way that a measuring voltage can be
applied across a single array at a given time using switches (such
as electronic switches).
[0037] Preferably, each electrode array of the device has an
approximately uniform electrode resistance distribution across the
entire array. By "uniform resistance distribution across the array"
is meant that when a measurement voltage is applied across the
electrode structures of the array, the electrode resistance at any
given location of the array is approximately equal to the electrode
resistance at any other location on the array. Preferably, the
electrode resistance at a first location on an array of the device
and the electrode resistance at a second location on the same array
do not differ by more than 30%. More preferably, the electrode
resistance at a first location on an array of the device and the
electrode resistance at a second location on the same array do not
differ by more than 15%. Even more preferably, the electrode
resistance at a first location on an array of the device and a
second location on the same array do not differ by more than 5%.
More preferably yet, the electrode resistance at a first location
on an array of the device and a second location on the same array
do not differ by more than 2%.
[0038] Preferred arrangements for electrode elements and gaps
between the electrodes and electrode buses in a given electrode
array are used to allow all cells, no matter where they land and
attach to the electrode surfaces and to contribute similarly to the
total impedance change measured for the electrode array. Thus, it
is desirable to have similar electric field strengths at any two
locations within any given array of the device when a measurement
voltage is applied to the electrode array. At any given location of
the array, the field strength is related to the potential
difference between the nearest point on a first electrode structure
of the array and the nearest point on a second electrode structure
of the array. It is therefore desirable to have similar electric
potential drops across the electrode elements and across the
electrode buses of a given array. Based on this requirement, it is
preferred to have an approximately uniform electrode resistance
distribution across the whole array where the electrode resistance
at a location of interest is equal to the sum of the electrode
resistance between the nearest point on a first electrode structure
(that is the point on the first electrode structure nearest the
location of interest) and a first connection pad connected to the
first electrode structure and the electrode resistance between the
nearest point on a second electrode structure (that is the point on
the first electrode structure nearest the location of interest) and
a second connection pad connected to the second electrode
structure.
[0039] Preferably, devices of the present invention are designed
such that the arrays of the device have an approximately uniform
distribution across the whole array. This can be achieved, for
example, by having electrode structures and electrode buses of
particular spacing and dimensions (lengths, widths, thicknesses and
geometrical shapes) such that the resistance at any single location
on the array is approximately equal to the resistance at any single
other location on the array. In most embodiments, the electrode
elements (or electrode structures) of a given array will have even
spacing and be of similar thicknesses and widths, the electrode
buses of a given array will be of similar thicknesses and widths,
and the electrode traces leading from a given array to a connection
pad will be of closely similar thicknesses and widths. Thus, in
these preferred embodiments, an array is designed such that the
lengths and geometrical shapes of electrode elements or structures,
the lengths and geometrical shapes of electrode traces, and the
lengths and geometrical shapes of buses allow for approximately
uniform electrode resistance distribution across the array.
[0040] In some preferred embodiments of impedance measurement
devices, electrode structures comprise multiple electrode elements,
and each electrode element connects directly to an electrode bus.
Electrode elements of a first electrode structure connect to a
first electrode bus, and electrode elements of a second electrode
structure connect to a second electrode bus. In these embodiments,
each of the two electrode buses connects to a separate connection
pad via an electrical trace. Although the resistances of the traces
contribute to the resistance at a location on the array, for any
two locations on the array the trace connections from the first bus
to a first connection pad and from the second bus to a second
connection pad are identical. Thus, in these preferred embodiments
trace resistances do not need to be taken into account in designing
the geometry of the array to provide for uniform resistances across
the array.
[0041] In preferred embodiments of the present invention, a device
for monitoring cell-substrate impedance has two or more electrode
arrays that share a connection pad. Preferably one of the electrode
structures of at least one of the electrode arrays of the device is
connected to a connection pad that also connects to an electrode
structure of at least one other of the electrode arrays of the
device. Preferably for at least two arrays of the device, each of
the two or more arrays has a first electrode structure connected to
a connection pad that connects with an electrode structure of at
least one other electrode array, and each of the two or more arrays
has a second electrode structure that connects to a connection pad
that does not connect with any other electrode structures or arrays
of the device. Thus, in preferred designs of a device there are at
least two electrode arrays each of which has a first electrode
structure that is connected to a common connection pad and a second
electrode structure that is connected to an independent connection
pad.
[0042] In some preferred embodiments of the present invention, each
of the electrode structures of an array is connected to an
electrode bus that is connected to one of the two or more
connection pads of the device via an electrically conductive trace.
In preferred embodiments, each of the two electrode structures is
connected to a single bus, such that each array connects to two
buses, one for each electrode structure. In this arrangement, each
of the two buses connects to a separate connection pad of the
substrate.
[0043] The electrically conductive traces that connect a bus with a
connection can be fabricated of any electrically conductive
material. The traces can be localized to the surface of the
substrate, and can be optionally covered with an insulating layer.
Alternatively the traces can be disposed in a second plane of the
substrate. Description of arrangements and design of electrically
conductive traces on impedance measurement devices can be found in
U.S. Pat. No. 7,470,533, herein incorporated by reference for all
disclosure on fabrication and design of electrically conductive
trace on substrates.
[0044] Appropriate electronic connection means such as metal clips
engaged onto the connection pads on the substrate and connected
printed-circuit-boards can be used for leading the electronic
connections from the connection pads on the devices to external
electronic circuitry (e.g. an impedance analyzer). Description of
the design of cell-substrate impedance devices and their
manufacture can be found in U.S. Pat. No. 7,470,533, herein
incorporated by reference for all description and disclosure of the
design, features, and manufacture of impedance device comprising
electrode arrays.
[0045] Descriptions of electrode arrays used for impedance
measurement that apply to the devices of the present invention are
also described in U.S. Pat. No. 7,470,533, herein incorporated by
reference for all disclosure relating to electrode arrays (or
structural units), electrode structures, electrode materials,
electrode dimensions, and methods of manufacturing electrodes on
substrates.
[0046] Preferred electrode arrays for devices of the present
invention include arrays comprising two electrode structures, such
as, for example, spiral electrode arrays and interdigitated arrays.
In some preferred devices of the present invention, electrode
arrays are fabricated on a substrate, in which the arrays comprises
two electrode structures, each of which comprises multiple
circle-on-line electrode elements, in which the electrode elements
of one structure alternate with the electrode elements of the
opposite electrode structure. Electrode arrays may be provided in
configurations, such as interdigitated, circle-on-line,
diamond-on-line, concentric, sinusoidal and castellated.
[0047] Preferably, the electrode elements (or electrode structures)
of an array of the present device of the present invention are of
approximately equal widths. Preferably the electrode elements (or
electrode structures) of an array of the present device of the
present invention are greater than 20 microns and less than 500
microns in width, more preferably from about 50 to about 300
microns in width.
[0048] Preferably, the electrode elements (or electrode structures)
of an array of the present device of the present invention are
approximately evenly spaced. Preferably, the gap between electrode
elements (or electrode structures) of an array of the present
device of the present invention is less than 100 microns and more
than 5 microns in width, more preferably from about 10 to about 80
microns in width.
[0049] Preferably, the device includes one or more
fluid-impermeable receptacles which serve as fluid containers or
wells. Such receptacles may be reversibly or irreversibly attached
to or formed within the substrate or portions thereof (such as, for
example, wells formed as in a microtiter plate). In another
example, the device of the present invention includes
microelectrode strips reversibly or irreversibly attached to
plastic housings that have openings that correspond to electrode
structure units located on the microelectrode strips. Suitable
fluid container materials comprise plastic, glass, or plastic
coated materials such as a ceramic, glass, metal, etc. Descriptions
and disclosure of devices that comprise fluid containers can be
found in U.S. Pat. No. 7,470,533, herein incorporated by reference
for all disclosure of fluid containers and fluid container
structures that can engage a substrate comprising electrodes for
impedance measurements, including their dimensions, design,
composition, and methods of manufacture.
[0050] In preferred embodiments, each electrode array on the
substrate of a device of the present invention is associated with a
fluid-impermeable container or receptacle, such as, for example, a
well. Preferably, the device of the present invention is assembled
to a bottomless, multiwell plastic plate or strip with a fluid
tight seal. The device is assembled such that a single array of the
substrate is at the bottom of a receptacle or well. Preferably,
each array of a device is associated with a well of a multiwell
plate. In some preferred embodiments, a multiwell device for
cell-substrate impedance measurement has "non-array" wells that are
attached to the substrate but not associated with arrays. Such
wells can optionally be used for performing non-impedance based
assays, or for viewing cells microscopically.
[0051] The design and assembly of multiwell impedance measurement
devices is described in U.S. Pat. No. 7,470,533, and also in U.S.
Pat. No. 7,192,752, both herein incorporated by reference for
disclosure of multiwell impedance measurement devices, including
their design, composition, and manufacture. A device of the present
invention preferably has between 2 and 1,536 wells and more
preferably between 4 and 384 wells. In some embodiments the device
includes 6 wells, 16 wells, 32 wells, 96 wells or 386 wells.
[0052] In some preferred embodiments, commercial tissue culture
plates can be adapted to fit a device of the present invention.
Bottomless plates may also be custom-made to preferred dimensions.
Preferably, well diameters are from about 1 millimeter to about 20
millimeters, more preferably from about 2 millimeters to about 8
millimeters at the bottom of the well (the end disposed on the
substrate). The wells can have a uniform diameter or can taper
toward the bottom so that the diameter of the container at the end
in contact with the substrate is smaller than the diameter of the
opposing end.
[0053] In the system for monitoring impedance of beating cells the
impedance analyzer engages connection pads of one or more
multi-well devices to measure impedance. In one embodiment of the
above system, the impedance analyzer is capable of measuring
impedance between 0.1 ohm and 10.sup.5 ohm in frequency range of 1
Hz to 1 MHz. The impedance analyzer is preferably capable of
measuring both resistance and reactance (capacitive reactance and
inductive reactance) components of the impedance. In a preferred
embodiment of the above system, the impedance analyzer is capable
of measuring impedance between 1 ohm and 10.sup.3 ohm in frequency
range of 1.00 Hz to 300 kHz.
[0054] In preferred embodiments the impedance analyzer is capable
of impedance measurements at millisecond time resolution. The
required or desired time resolution may vary depending on the
excitation cycle of the excitable cell. Excitable cells having
shorter excitation cycles would tend to require faster time
resolution. In some embodiments 500 millisecond time resolution is
sufficient, such that at least two consecutive impedance
measurements are between about 300 milliseconds and about 500
milliseconds apart. In preferred embodiments, impedance measurement
at millisecond time resolution includes at least two consecutive
impedance measurements less than 100 milliseconds apart. In some
instances the at least two consecutive impedance measurements are
less than 50 milliseconds or less than 40 milliseconds apart. In
some instances the at least two consecutive impedance measurements
are less than 20 milliseconds apart. In some instances at least two
consecutive impedance measurements are less than 10 milliseconds
apart. In some instances millisecond time resolution includes two
consecutive impedance measurements between 1 millisecond and 5
milliseconds, between 5 milliseconds and 10 milliseconds, between
10 milliseconds and 20 milliseconds, between 20 milliseconds and 40
milliseconds, or between 40 milliseconds and 50 milliseconds apart.
In some instances millisecond time resolution includes at least two
consecutive impedance measurements between 50 milliseconds and 100
milliseconds apart. In some instances millisecond time resolution
includes at least two consecutive impedance measurements between
100 milliseconds and 150 milliseconds or between 150 and 300
milliseconds apart.
[0055] With millisecond time resolution for impedance measurement,
it becomes possible to resolve individual beating cycles of
cardiomyocytes cultured on electrodes. Whilst theoretically one
needs at least two data points for each beating cycle, in practice
more than 2 data points are needed for each beating cycle. For
example, if cells have a beating rate of 60 beats per minute, i.e.,
one beat per second. It would be preferred to have a time
resolution of at least 200 milliseconds so that each beating cycle
consists of 5 data points. More preferably, the measurement time
resolution is 100 milliseconds. Still more preferably, the time
resolution is 50 milliseconds or less.
[0056] One skilled in the art will understand that the
cell-substrate impedance measurement or monitoring system with
millisecond time resolution can be used to efficiently and
simultaneously perform multiple assays by using circuitry of the
device station to digitally switch from recording from measuring
impedance over an array in one well to measuring impedance over an
array in another well. Similarly, groups of wells may be monitored
simultaneously and switched between designated groups. In one
embodiment of the above system, the system under software control
is capable of completing an impedance measurement for an individual
well at a single frequency within milliseconds, such as less than
100 milliseconds, less than 40 milliseconds, less than 20
milliseconds, less than 10 milliseconds or between 1 millisecond
and 40 milliseconds. In some embodiments the user may choose the
frequency of measurement for millisecond time resolution.
[0057] A multiple-well cell-substrate impedance measuring device in
a system of the present invention can be any multiple-well
cell-substrate impedance measuring device in which at least two of
the multiple wells comprise an electrode array at the bottom of the
well, and in which at least two of the multiple wells comprise an
electrode array are individually addressed. In one embodiment of
the above system, the multi-well device takes the form of a
specialized microtiter plate which has microelectronic sensor
arrays integrated into the bottom of the wells.
[0058] A device used in a system of the present invention, when
connected to an impedance analyzer, can measure differences in
impedance values that relate to cell behavior. For example, a
cell-substrate impedance measuring device used in a system of the
present invention can measure differences in impedance values when
cells are attached to the electrode array and when cells are not
attached to the electrode array, or can detect differences in
impedance values when the number, type, activity, adhesiveness, or
morphology of cells attached to the electrode-comprising surface of
the apparatus changes. Further, by using millisecond time
resolution differences in impedance may be detected or monitored
that relate to excitation-contraction coupling, including the
beating of cardiomyocytes or stem cells differentiating into
cardiomyocytes, and the signaling between neurological cells.
Impedance monitoring of the excitation cycle of excitable cells may
be determined and monitored before, during or after adding a test
compound, which is suspected of affecting the excitation cycle.
Thus, by monitoring the excitation cycle of the excitable cell
before, during or after adding a test compound the system provides
data corresponding to the potential affect of the compound on the
cardiovascular system, the heart, the nervous system, and the like.
In some embodiments monitoring the excitation cycle of the cell
before, during or after adding a compound provides cardiotoxicity
data useful in drug screening.
[0059] In some embodiments a device station or electromechanical
apparatus or assembly capable of interfacing multiwell devices can
include one or more platforms or one or more slots for positioning
one or more multiwell devices. The one or more platforms or one or
more slots can comprise sockets, pins or other devices for
electrically connecting the device to the device station. The
device station or electromechanical apparatus or assembly capable
of interfacing multiwell devices preferably can be positioned in a
tissue culture incubator during cell impedance measurement assays.
It can be electrically connected to an impedance analyzer and
computer that are preferably located outside the tissue culture
incubator.
[0060] The device station or electromechanical apparatus or
assembly capable of interfacing multiwell devices includes
electronic circuitry that can connect to the impedance monitoring
device and an impedance analyzer and electronic switches that can
switch on and off connections to each of the two or more electrode
arrays of the multiwell devices used in the system. The switches of
the device station or electromechanical apparatus or assembly
capable of interfacing multiwell devices are controlled by a
software program, each of which has been improved to provide
millisecond time resolution. The software program directs the
device station to connect arrays of the device to an impedance
analyzer and monitor impedance from one or more of the electrode
arrays. During impedance monitoring, the impedance analyzer can
monitor impedance at one frequency or at more than one frequency.
Preferably, impedance monitoring is performed at more than one time
point for a given assay, and preferably, impedance is monitored
using at least two time points. The device station can connect
individual arrays of a device to an impedance analyzer to monitor
one, some, or all of the arrays of a device for a measurement time
point. In some preferred embodiments of the present invention, the
device station software is programmable to direct impedance
monitoring of any of the wells of the device that comprise arrays
at chosen time intervals.
[0061] The software of the impedance monitoring system can also
store and display data. Data can be displayed on a screen, as
printed data, or both. Preferably the software can allow entry and
display of experimental parameters, such as descriptive information
including cells types, compound concentrations, time intervals
monitored, etc. Further, since a plurality of beating parameters
are obtained using the beating measurements, software provides
menus to select one or more of the beating parameters for
analysis.
[0062] The software, termed RTCA CARDIO SOFTWARE (ACEA Biosciences
Inc., San Diego, Calif.), also permits fast analysis of beating
parameters. Thus, after obtaining impedance measurements the
software can calculate or determine from the impedance measurement
a plurality of beating parameters such as beating rate, beating
amplitude, rising time, falling time, beating period, IBD10, IBD50,
IBD90, rising slope, falling slope, normalized beating rate,
normalized beating amplitude, beating pattern similarity and
beating rhythm irregularity, and perform subsequent statistics,
such as average and standard deviation and further supply IC50 or
EC50 values for dose-response testing.
Determining Cell Index from Impedance or Optical Measurements
[0063] Although raw impedance values or raw values obtained from
optical sensors may be used as beating measurements in preferred
embodiments the raw impedance values are converted to cell index
values or delta cell index values for comparison or further
derivation into beating parameters as discussed below. Information
regarding how to calculate a cell index, cell change index,
normalized cell index, and delta cell index may be found in U.S.
patent application Ser. No. 12/435,569, U.S. patent application
Ser. No. 11/903,454, and U.S. Pat. No. 7,470,533, the contents of
which are herein incorporated by reference with respect to the cell
index, cell index number, cell change index, and cell change index
number. However a briefly summary is provided.
[0064] The cell index obtained for a given well reflects: 1) how
many cells are attached to the electrode surfaces in this well, and
2) how well cells are attached to the electrode surfaces in the
well. In this case, a zero or near-zero "cell index or cell number
index" indicates that no cells or very small number of cells are
present on or attached to the electrode surfaces. In other words,
if no cells are present on the electrodes, or if the cells are not
well-attached onto the electrodes the cell index equals 0. A higher
value of "cell index" or "cell number index" indicates that, for
the same type of the cells and cells under similar physiological
conditions, more cells are attached to the electrode surfaces. Thus
cell index is a quantitative measure of cell number present in a
well. A higher value of "cell index" may also indicate that, for
the same type of the cells and the same number of the cells, cells
are attached better (for example, cells spread out more, or cell
adhesion to the electrode surfaces is stronger) on the electrode
surfaces.
[0065] Non-limiting examples for determining cell index follow.
Cell index can be calculated by: at each measured frequency,
calculating the resistance ratio by dividing the measured
resistance (when cells are attached to the electrodes) by the
baseline resistance; finding or determining the maximum value in
the resistance ratio over the frequency spectrum; and subtracting
one from the maximum value in the resistance ratio. In another
variation, cell index is determined by: at each measured frequency,
calculating the resistance ratio by dividing the measured
resistance (when cells are attached to the electrodes) to the
baseline resistance; finding or determining the maximum value in
the resistance ratio over the frequency spectrum; and taking a
log-value (e.g., based on 10 or e=2.718) of the maximum value in
the resistance ratio. In another variation, cell index is
determined by: at each measured frequency, calculating the
reactance ratio by dividing the measured reactance (when cells are
attached to the electrodes) to the baseline reactance; finding or
determining the maximum value in the reactance ratio over the
frequency spectrum; and subtracting one from the maximum value in
the resistance ratio. In still another variation, cell index can be
calculated by: at each measured frequency, calculating the
resistance ratio by dividing the measured resistance (when cells
are attached to the electrodes) to the baseline resistance; then
calculating the relative change in resistance in each measured
frequency by subtracting one from the resistance ratio; and then
integrating all the relative-change value.
[0066] It is worthwhile to point out that it is not necessary to
derive such a "cell index" for utilizing the impedance information
for monitoring cell conditions over the electrodes. Actually, one
may choose to directly use impedance values (e.g., at a single
fixed frequency; or at a maximum relative-change frequency, or at
multiple frequencies) as an indicator of cell conditions.
[0067] A "normalized cell index" at a given time point is
calculated by dividing the cell index at the time point by the cell
index at a reference time point. Thus, the normalized cell index is
1 at the reference time point. Normalized cell index is cell index
normalized against cell index at a particular time point. In most
cases in the present applications, normalized cell index is derived
as normalized relative to the time point immediately before a
compound addition or treatment. Thus, normalized cell index at such
time point (immediately before compound addition) is always unit
one for all wells. One possible benefit for using such normalized
cell index is to remove the effect from difference in cell number
in different wells. A well having more cells may produce a larger
impedance response following compound treatment. Using normalized
cell index, it helps to remove such variations caused by different
cell numbers.
[0068] A "delta cell index" at a given time point is calculated by
subtracting the cell index at a standard time point from the cell
index at the given time point. Thus, the delta cell index is the
absolute change in the cell index from an initial time (the
standard time point) to the measurement time.
[0069] The time-dependent cellular response (including
cardiotoxicity response) may be analyzed by deriving parameters
that directly reflect the changes in cell status. For example, time
dependent cellular response may be analyzed by calculating the
slope of change in the measured impedance responses (that is
equivalent to the first order derivative of the impedance response
with respect to time, impedance response here can be measured
impedance data or derived values such as cell index, normalized
cell index or delta cell index). In another example, the
time-dependent cellular responses (including cardiotoxic responses)
may be analyzed for their higher order derivatives with respect to
time. Such high order derivatives may provide additional
information as for how cells responding to different compounds and
as for the mechanisms of compound action.
[0070] The use of cell index together with millisecond time
resolution impedance monitoring is demonstrated in FIGS. 2-6, which
depict high resolution images of the cardiomyocyte beating cycle
and pattern shifting in response to administration of cardiotoxic
compounds. While cell index permits graphically depicting the
beating cycle, still more detailed analysis of beating parameters
reveals improvements to cardiomyocyte population analysis.
Beating Parameters Derived from Millisecond Time Resolution
Measurements or Cell Index
[0071] While impedance measurements, optical measurements and cell
index values can provide valuable information about a cell
population, a variety of beating parameters are established which
further assist in the analysis of a beating cardiomyocyte, a
beating cell or cell population differentiating into a beating cell
population. Further, the beating parameters permit comparisons
before and after treatment with a compound, typically a test
compound, to assess its effect or predicted effect on a
cardiomyocyte cell population and the like. As such, the beating
parameters may be used to establish or confirm safety of a compound
or provide further insight as to the potential mechanism of action
of a compound, such as its effect on stem cell differentiation to
or away from a cardiomyocyte cell type or characteristic. Further,
by providing a compound at various concentrations its dose response
can be studied. The below beating parameters have been found useful
in assessing potential effects on cardiomyocytes and thus each
alone or in combination can be used to assess potential risk of
compound based therapies.
[0072] In one approach beating measurements are used to determine
beating cycle peaks associated with a cell. Beating itself
corresponds to the excitation-contraction coupling of the cells.
Turning to FIG. 7, beatings are defined as a sequence of Positive
Peaks (+P as labeled) and Negative Peaks (-P as labeled). The value
of these Positive Peaks and Negative Peaks and the corresponding
time periods determine the beating characteristics, which reveal
the status of the cardiomyocyte population. For example, in FIG. 7,
a Positive Peak may correspond to the contraction of
cardiomyocytes, whilst the return of measurement values to baseline
and to negative peak may correspond to the relaxation of
cardiomyocytes.
[0073] As an example, time dependent impedance values or cell index
values for a well are analyzed by deriving their first order
derivatives and second order derivatives using numerical methods.
The beating cycle peaks are those data points where the first order
derivatives of impedance values or cell index values are zero or
close to zero in absolute value. If the beating cycle peak is a
positive peak (i.e. peak corresponds to a maximum value in measured
impedance or cell index over the beating cycle), then the peak
would correspond to data points where the second order derivatives
of the impedance values or cell index values are negative and where
the first order derivatives of the impedance values or cell index
values are zero or close to zero in absolute value. If the beating
cycle peak is a negative peak (i.e. peak corresponds to a minimum
value in measured impedance or cell index over the beating cycle),
then the peak would correspond to the data points where the second
order derivatives of the impedance values or cell index values are
positive and where the first order derivatives of the impedance
values or cell index values are zero or close to zero in absolute
value.
[0074] In yet another approach, the method for searching for and
identifying "positive peaks" and "negative peaks" may involve the
use and modification of various mathematical algorithms, e.g., the
Douglas-Peucker algorithm. The Douglas-Peucker algorithm is an
algorithm for reducing the number of points in a curve that is
approximated by a series of points. Based on the required maximum
distance between on the original curves and on the simplified
curves, the Douglas-Peucker algorithm could also be adopted to
identify positive peaks and negative peaks in time-dependent data
point series for impedance values and/or cell index vales.
[0075] In another approach, a method of determining a beating cycle
peak is to search for the data point where the trend of the data
changes direction from "increasing" to "decreasing" with time (for
a positive peak), or from "decreasing" to "increasing" (for a
negative peak). After the identification of the beating cycle
peaks, the impedance or cell index values at such peak time points
provide the magnitude or amplitude of the beating cycle peaks.
[0076] After determining the beating cycle peaks, various methods
can be used to calculate the beating rate. A beating rate parameter
is generally provided as beatings per minute. In a positive peak
counting approach, the number of positive peaks is determined over
a given time interval and converted to the desired unit, preferably
beats per minute. Similarly, in a negative peak counting approach
the number of negative peaks is determined over given time interval
and converted to the desired unit. As an example, if there are 2
peaks in a one second interval, then the beating rate would be 2
beats per second, or 120 beats per minute. In still another
approach, beating rate is calculated by determining the time period
between a series of two or more positive peaks or between a series
of two or more negative peaks. That is, in this approach a unit of
time (e.g., 1 minute) is divided by the time period between two
adjacent peaks. For example, if two adjacent peaks are separated by
500 milliseconds, then the beating rate would be 120 beats per
minute. In a time interval comprising multiple positive or negative
peaks, the beating rate could be determined by the following
method. Take positive peaks as an example, the time periods between
every pairs of two adjacent positive peaks are calculated. Then the
beating rate could be calculated in two ways. The first method is
to divide a unit of time (e.g., 1 minute) by the average of the
time periods between all two-adjacent positive peaks in the given
time interval. The second method is to calculate the corresponding
beating rates based on each pair of two adjacent positive peaks and
then to average of the adjacent-peaks-derived beating rates.
[0077] To further assist in comparison, beating rates can also be
normalized. Determining a normalized beating rate is achieved by
dividing the beating rate at a selected data analysis time by a
beating rate at a normalization time. Thus a beating rate identical
to that at the normalization time would be defined as 1.
Normalizing beating rates can provide a more clear indication of
whether and to what degree a change in beating rate occurs. For
example, normalization time corresponds to the time point of
measurement immediately prior to a compound treatment of the
cardiomyocytes. Thus, normalized beating rates could provide clear
information as for what the effect of the compound has on the
beating rates. A normalized beating rate close to 1 or equal to 1
means that the compound does not have much effect on the beating
rate, A normalized beating rate smaller than 1 means that the
compound may result in reduction in the beating rates of
cardiomyocytes. A normalized beating rate larger than 1 means that
the compound may cause an increase in the beating rates of
cardiomyocytes. Normalized beating rates could be derived for
beating rates calculated using different methods such as
peak-counting derived beating rates and beating peak periods
derived beating rates.
[0078] Beating amplitude is a parameter used in some embodiments to
describe or correspond to the intensity of the peak, which may
reflect the extent of contraction or relaxation of cardiomyocytes
during a beating cycle. Determining beating amplitude can involve a
whole peak approach, which could be determined by the difference
between a negative peak and the following positive peak as shown in
FIG. 7. For example, in FIG. 7 the beating amplitude is shown as
the difference in cell index between a Negative Peak to the
following adjacent Positive Peak (Amp-1, Amp-2, Amp-3, . . . ,
Amp-m). In another approach, beating amplitude for a positive peak
is the difference between a determined baseline to a positive peak.
In still another approach beating amplitude for a negative peak is
the difference between a determined baseline to a negative peak. An
exemplary baseline is shown in FIG. 7.
[0079] Thus, for a single beating cycle, one could define or
identify different types of amplitude (or an amplitude) of the
beating-cycle peaks, including the amplitude of positive peak, the
amplitude of negative peak and the amplitude of the whole peak in a
cycle. From the measured data point series, a baseline value, which
may theoretically correspond to the value when the cardiomyocytes
are at their fully relaxation status, could be determined or
identified from the measured data values in a time series. The
amplitude of a positive peak is the impedance value or cell index
value or other measurement value at the positive-peak time point
subtracted by the baseline value. The amplitude of a negative peak
is the impedance value or cell index value or other measurement
value at the negative-peak time point subtracted by the baseline
value. The amplitude of whole peak is the difference in the
impedance value or cell index value or other measurement value
between positive-peak time point and negative-peak time point.
[0080] Whilst the above paragraph discusses different types of the
amplitudes of a single beating cycle, for a time period including
multiple beating cycles, one could determine the average and
standard deviations (or standard errors) of the positive-peak
amplitude, the negative-peak amplitude and whole-peak
amplitude.
[0081] Beating amplitude can also be normalized as a normalized
amplitude. A normalized amplitude is the amplitude at a selected
data analysis time divided by the amplitude at the normalization
time point. A beating amplitude identical to that at the
normalization time would be defined as 1. Thus, the normalized
amplitude reveals differences, such as an increase or decrease in
the amplitude or intensity of a beat compared to a referenced
amplitude. For example, normalization time corresponds to the time
point of measurement immediately prior to a compound treatment of
the cardiomyocytes. Thus, normalized beating amplitudes could
provide clear information as for what the effect of the compound
has on the beating amplitudes. A normalized beating amplitude close
to 1 or equal to 1 means that the compound does not have much
effect on the beating amplitude. A normalized beating amplitude
smaller than 1 means that the compound may result in reduction in
the beating amplitudes of cardiomyocytes. A normalized beating
amplitude larger than 1 means that the compound may cause an
increase in the beating rates of cardiomyocytes.
[0082] Normalized beating amplitude could be derived for all three
types of beating amplitudes, i.e. positive-peak based amplitude,
negative-peak based amplitude and whole-peak amplitude.
[0083] In some instances, it is more useful to consider a beating
amplitude that is less than the difference between the positive
peak and negative peak or baseline. For example, in may be
desirable to consider a portion of the amplitude, such as a 10%
amplitude, 20% amplitude, 50% amplitude, 80% amplitude, 90%
amplitude and the like. This may be particularly preferred when
assessing other parameters together with beating amplitude such as
when considering a rising slope or an IBD10, IBD50, IBD90 and the
like.
[0084] Rising time T.sub.r is a parameter which provides the amount
of time to travel the rising slope between the negative peak and
the positive peak (or between the baseline and the positive peak).
Typically, rising time is provided as an average over many beatings
and is converted to time in the unit of seconds for analysis or
comparison. In some embodiments rising time is calculated as the
time needed to reach a positive peak or maximum amplitude from a
negative peak or a baseline. Referring to FIG. 8 as an example, in
other instances, rising time T.sub.r is calculated between data
points positioned below the positive peak and above the negative
peak (or below the positive peak and above the baseline). That is,
since the slope of the beating curve changes dramatically at the
peaks, it is often preferred to consider the rising time between
20% amplitude and 80% amplitude for comparison. Thus, when
determining rising time it may be necessary to first determine the
beating amplitude then determine suitable comparison points such as
20% amplitude, 50% amplitude, 80% amplitude and the like. By one
example as illustrated in FIG. 8, rising slope can be calculated by
the dividing the cell index change from 20% amplitude to 80%
amplitude by the time it takes as follows:
Rising Slope=(Amp.sub.80-Amp.sub.20)/T.sub.r
[0085] Depending on specific application, it is possible that we
may define other rising time and/or rising slopes, using different
percentage amplitude points. For example, rising slope may be
defined as:
Rising Slope=(Amp.sub.90-Amp.sub.10)/T.sub.r
[0086] Falling time T.sub.f is a beating parameter which provides
the amount of time to travel the falling slope between a positive
peak or a point of maximum amplitude to a negative peak or a
baseline. Falling time T.sub.f can be provided as an average over
many beatings and is generally converted to time in the unit of
seconds for analysis or comparison. In some embodiments falling
time T.sub.f is calculated as the time needed to reach a negative
peak or baseline from a positive peak; however, referring again to
FIG. 8 as an example, in other instances, falling time T.sub.f is
calculated between data points positioned below the positive peak
and above the negative peak. That is, since the slope of the
beating curve changes dramatically at the peaks, it is often
preferred to consider the falling time between 80% amplitude (or
90% amplitude) and 20% amplitude (or 10% amplitude) for comparison.
Thus, when determining falling time it may be necessary to first
determine the beating amplitude then determine suitable comparison
points such as 10% amplitude, 20% amplitude, 50% amplitude, 80%
amplitude, 90% amplitude and the like. By way of example, falling
slope can be calculated by dividing the cell index change from 80%
amplitude to 20% amplitude by the time it takes as follows:
Falling Slope=(Amp.sub.80-Amp.sub.20)/T.sub.r
[0087] Depending on specific application, it is possible that we
may define other rising time and/or rising slopes, using different
percentage amplitude points. For example, falling slope may be
defined as:
Falling Slope=(Amp.sub.90-Amp.sub.10)/T.sub.r.
[0088] IBD50 provides the time window between time points where the
beating signals attains 50% amplitude. IBD50 is particularly useful
when studying the effect of the compounds on the beating cycle of
cardiomyocytes. For example, if a compound, through various
mechanisms, results in prolongation of contraction phase of the
beating cycle of the cardiomyocytes, then IBD50 derived from the
beating cycle waveform may be increased as a result of the
treatment of cardiomyocytes with this compound. In another example,
if a compound results in certain pro-arrhythmia effects in
cardiomyocytes (see FIG. 3 as an example where E4031 induced
arrhythmia effects), then IBD50 derived from the beating cycle
waveform may also be significantly altered. Similarly, other time
windows can be used instead to assess similar pro-arrhythmia
effects at different percentage of the amplitude such as 10%
amplitude and 90% amplitude to give IBD10 and IBD90. Time between
following IBD10, IBD50 or IBD90 measurements are designated as
T.sub.IBD10, T.sub.IBD50, and T.sub.IBD90. An exemplary T.sub.IBD50
parameter is shown in FIG. 8.
[0089] Beating period (also referred to as "beating cycle") is a
parameter which provides the time period between two positive
peaks, two negative peaks or can be the time period between a
positive peak and a negative peak. The beating period can be used
to identify changes in beating rate or can be used as a defined
period for comparison of other parameters, such as differences in
amplitude, rising time, falling time and the like. In FIG. 7,
within one Sweep Duration, the number of Positive Peaks is m (+P1,
+P2, +P3, . . . , +Pm) and the number of Negative Peaks is n (-P1,
-P2, -P3, . . . , -Pn). The time between two adjacent individual
Positive Peaks (or/and two adjacent individual Negative Peaks) is
defined as beating period. For example, the beating period based on
the Positive Peaks is T.sub.+P1, T.sub.+P2, T.sub.+P(m-1) and the
beating period based on the Negative Peaks is T.sub.-P1, T.sub.-P2,
. . . , T.sub.-P(n-1).
[0090] Rising slope is a parameter corresponding to the slope that
occurs during a specified time period when the measured impedance
values or cell index values or other measurement/derived values
increase with time. That is, the rising slope is a slope between a
negative peak and an adjacent baseline point (which is after the
negative peak in time), or between certain baseline point and an
adjacent positive peak (which is after the baseline point in time),
or between a negative peak and its immediately adjacent positive
peak (which is after the negative peak in time). For a single beat,
one could determine rising slopes based on different definitions as
described in the previous sentence. The rising slope may be an
average between multiple slopes across multiple beats.
[0091] Falling slope is a parameter that corresponds to the slope
that occurs during a specified time period when the measured
impedance values or cell index values or other measurement/derived
values decrease with time. Thus, the falling slope is a slope
between a positive and an adjacent baseline point (which is after
the positive peak in time), or between certain baseline point and a
negative peak (which is after the baseline point in time), or
between a positive peak and its immediately adjacent negative peak
(which is after the positive peak in time). For a single beat, one
could determine falling slopes based on different definitions as
described in the previous sentence. The falling slope may be an
average between multiple slopes across multiple beats.
[0092] Beating pattern similarity is a parameter derived to
quantify the degree of the similarity between the beating waveforms
between two different time intervals. For any given time interval,
the beating pattern is shown as the beating curves comprised of a
number of measurement values (impedance values, cell index values
or other values) across a number of time points during the time
interval. Beating patterns at two time intervals may be compared
numerically, such as by comparison between determined parameters
for the beating curves at these two time intervals or patterns may
be compared through the comparison of the beating curves. When
comparing curves it may desirable to align curves to match an
initial positive peak or initial negative peak. Aligning curves may
also use a variety of curve algorithms, which identify distances or
shifts between curves.
[0093] In one embodiment, the beating pattern similarity is derived
as a parameter to compare the determined parameters for the beating
curves at two time intervals. For example, one may compare the
beating rates BR.sub.1 and BR.sub.2 at the two time intervals. An
example of the beating pattern similarity is given as:
Beating pattern
similarity=(2*BR.sub.1*BR.sub.2)/(BR.sub.1*BR.sub.1+BR.sub.2*BR.sub.2)
[0094] With this above example, the beating pattern similarity is
one (the highest value) when the beating rates at the two time
intervals are the same. When the beatings rates differ at two time
intervals, the beating pattern similarity would be less than 1. The
more the beating rates differ, the smaller the beating pattern
similarity value.
[0095] In a preferred embodiment, however, the beating pattern
similarity is derived as a parameter to directly compare the
beating curves at the two time intervals. The idea of the beating
pattern similarity should possess such properties that the value
for beating pattern similarity is large when the two beating curves
are similar, and the value for beating pattern similarity is small
when the two beating curves are not similar. There are multiple
methods for deriving such beating pattern similarity values.
[0096] In one method, as briefly mentioned above, for comparing the
beating curves at two time intervals (assuming the same measurement
time resolutions for the measured data points), it may desirable to
align curves to match an initial positive peak or initial negative
peak. After aligning the initial peaks, an "AND" operation is
performed on the time points for the two beating curves so that the
overlapping time points on the two beating curves are kept whilst
non-overlapping time points on either one of the beating curves are
discarded. Thus, the remaining, overlapping data points on the two
beating curves are of the same number and it is possible to readily
define a distance to describe whether the two beating curves are
similar. For example, the beating pattern similarity could be the
correlation coefficient between the two data series in the
remaining portions of the two beating curves. Clearly, the more
similar the two curves, the larger the correlation coefficient
(i.e., the larger the beating pattern similarity value is). In
another example, the beating pattern similarity could simply be
certain mathematically-defined-distance (e.g. Euclidean distance)
between two data series in the remaining portion of the two beating
curves. Note that if the measurement time resolutions differ
between the measured data points, additional time points may be
artificially inserted into the beating curves with missing time
points after mathematically interpolation of the values for such
added time points based on other measured data points. With this
operation, the two beating curves would have the same time
resolutions.
[0097] In another method for comparing the beating curves at two
time intervals (again, assuming the same measurement time
resolutions for the measured data points), one would take the
beating curve (out of the two) with the shorter time duration. If
the time-shorter beating curve comprises more than half of the data
points of the other beating curve, then some last data points from
the shorter beating curve are removed to form a "base-curve" so
that the number of the remaining data points in the shorter beating
curve is half of the number of the data points in the other beating
curve. Then a number of correlation coefficients would be
determined where each correlation coefficient corresponds to the
base-curve aligned to one continuous segment (comprising of the
same data point number as the base-curve) of the other beating
curve. For example, the first correlation coefficient is determined
between the data series of the base curve and the data series of
first half of the other beating curve (starting from the first data
point). The second correlation coefficient is determined between
the data series of the base curve and the data series from the
other beating curve with starting point being the second data
point. The last correlation coefficient is determined between the
data series of the base curve and the data series from the second
half of the other beating curve ending with the last data point.
Finally, the beating pattern similarity is determined as the
maximum value of all the correlation coefficients.
[0098] There may be other methods or algorithms that could be used
for deriving beating pattern similarities. Beating pattern
similarity could be used to analyze the effect of a compound on the
beating pattern of cardiomyocytes. The beating curves from two time
intervals are compared. For analyzing the effect of a compound, one
time interval corresponds to the time period before compound
treatment whilst the other time interval corresponds to the time
period after compound treatment. The beating pattern similarity has
an advantage over other parameters in comparing compound's effect
on the cardiomyocytes. The advantage is that it could include or
summarize all the effects due to the compound, i.e. the effects on
the beating rates, beating waveform shapes or beating amplitudes
etc could all be included into the single parameter of the beating
pattern similarity.
[0099] Beating rhythm irregularity (BRI) is a parameter which
identifies changes in beating rate or changes between peak periods
for a beating curve over a time interval. Beating rhythm
irregularity is also referred to as a beating rate irregularity
index. If the beating rate or the beating peak period does not
change with time, then the beating rhythm is regular and the
parameter of the beating-rhythm-irregularity should be small, i.e.
being zero or close to zero. On the other hand, if the beating rate
or beating period does change with time, then beating rhythm is
irregular and the parameter of the beating rhythm irregularity
should have a large value. As one requirement, the parameter of the
beating rhythm irregularity should be able to identify the
arrhythmic beating of cardiomyocytes. Thus, the
beating-rhythm-irregularity should attain a large value for the
beating curves of cardiomyocytes if the cardiomyocytes exhibit
arrhythmic beating. There are multiple methods for calculating the
beating rhythm irregularity for a beating curve over a time
interval. For example, the positive peak periods for
each-adjacent-positive-peak-pair are calculated for the beating
curve in the given time interval. Then the average and standard
deviation of such multiple positive peak periods are calculated.
The beating rhythm irregularity can be calculated by dividing the
standard deviation of the positive peak periods by the average. In
another example, the negative peak periods for each adjacent
negative peak pair are calculated for the beating curve in the
given time interval. Then the average and standard deviation of
such multiple negative peak periods are calculated. The beating
rhythm irregularity can be calculated by dividing the standard
deviation of the negative peak periods by the average.
[0100] When the cardiomyocytes exhibit irregular beating, the
beating curves may comprise some regular beating peaks (positive or
negative) with regular amplitudes and some irregular beating peaks
with somewhat smaller (/or larger) amplitudes or with somewhat
different beating waveforms (e.g., the impedance value or cell
index value or other measurement value does not return to baseline
level after a beating peak). Thus, number or presence of the
irregular positive (or negative) beating peaks could be used as an
indicator for beating rhythm irregularity. Such regular and
irregular beats can be determined and the ratio between the
irregular and the regular beats, i.e. irregular to regular beats
ratio, can be used to assess the pro-arrhythmia effects of the test
compound. If there are no pro-arrhythmia effects, such ratio should
be very small and close to zero.
[0101] The skilled artisan will appreciate compounds may be added
to cardiomyocyte populations to test for potential changes or
effects in beating parameters. Changes may include an increase or
decrease in at least one of the beating parameters. The change may
include an increase in beating rhythm irregularity if the compound
causes an irregular beating in cardiomyocytes. If a compound
results in a significant effect on the beating of cardiomyocytes,
then beating pattern similarity between the beating curves before
and after the compound treatment may be small.
[0102] In some instances beating measurements are continually
monitored and a beating parameter is calculated for a single
beating cycle within a time period for comparison. For example, the
calculation for a beating parameter such as an amplitude can be
performed for a single beating cycle. Further, a beating parameter
from a single beating cycle (i.e. single beating peak) may provide
a suitable control for later determined beating parameters. Due to
the fact that single beating cycle may take a short time to
complete, beating parameter for a single beating cycle (i.e. single
beating peak) is sometimes referred as beating parameter at a
single time point.
[0103] Frequently, it may be desirable analyze one or more
parameters within a single beating period (i.e. for a single
beating cycle). From a single beating cycle (single beating period)
a variety of beating parameters can be assessed including beating
amplitude, rising or falling time, rising and falling slope and
IBD50 and the like. Consideration within a beating period may also
be particularly useful for use as an initial beating period before
the addition of a test compound to establish control beating data
for later comparison. The single beating period approach may also
be of interest if an irregularity is detected, such as an irregular
positive peak, negative peak, slope between peaks and the like. For
instance, an irregularity may be temporal and thus decay over time,
which if limited to studying across multiple beating periods could
be averaged out and thus not be identified as statistically
significant. Thus, if such an irregularity is identified, analysis
of the corresponding beating period may reveal significant
differences compared to control. Still further, analysis of the
single beating period may reveal a correlation to another change or
shift in beating parameter not apparent through multi-beating
period analysis.
[0104] In another approach one or more of the selected parameters
are determined or calculated across a time period that includes
multiple beating periods (i.e. multiple beating cycles). One or
more parameters can be determined for each of the desired multiple
beating periods. This approach may be used to monitor consistency
over time or as a continuous scanning technique to identify
potential periods of irregularity for further single beating period
analysis. The approach may also be used to develop average values
for comparison to test compounds.
[0105] While the methods permit the study of cardiomyocyte beating
itself including, changes in beating in response to cardiomyocyte
or stem cell differentiation and the like, the methods are also
useful to assess the effect of one or more test compounds on a
cardiomyocyte cell population. Such assays may help predict
potential cardiotoxic effects of compounds, effect on cell
differentiation or growth and the like. The skilled artisan will
appreciate parameters for cell populations treated with test
compounds may be compared to control treated populations, such as
vehicle control, and the resulting parameters compared to assess
for differences. Differences in beating parameters may be
indicative of cardiotoxicity or other effects.
[0106] In another embodiment, a dose response curve (DRC) of a test
compound is determined. A dose response curve reveals changes to
beating parameters in response to difference in concentrations of
test compound. Preferably, selected parameters are calculated from
beating measurements that are measured from wells having different
concentrations of the same test compound for a period of time where
beating signals are continuous. Preferably, for each concentration,
one value is calculated for the selected parameter for certain time
points after addition of test compound. Then a concentration
dependent dose response curve of the selected parameter is fitted
to a non-linear, sigmoidal-dose-response equation to derive EC50
(or IC50) values. An example of a sigmoidal dose-response curve is
as follows:
Y=Bottom+(Top-Bottom)/(1+10 (Log EC.sub.50-X))
[0107] An example of a sigmoidal dose-response with variable slop
is as follows:
Y=Bottom+(Top-Bottom)/(1+10 ((Log EC.sub.50-X)*Hill Slope))
[0108] The derived IC50/EC50 values for different compounds could
be used to predict, analyze, or compare the different compounds'
cardiotoxicity or different compounds' effect on beating of
cardiomyocytes. Like IC50 or EC50 derived from other assays, a
compound with lower IC50/EC50 values may indicate that the compound
is more potent in inducing cardiotocixicity effect or causing an
effect on beating of the cardiomyocytes than the compound with
higher IC50/EC50 values.
[0109] To further demonstrate the use of the above parameters,
modulators of ion channel and non-ion channel proteins and targets
as well as modulators of neuro-hormonal machinery were tested in
both time and dose-dependent manner (see for example FIGS. 3A-C and
FIGS. 4A-C). Using various parameters such as beating rate,
amplitude and IBD50, we have confirmed that the half maximal
concentrations obtained for the different compounds are within the
sensitivity range of patch clamp recording and other
electrophysiological methodologies (data not shown). Importantly,
we have also shown that certain classes of compounds such as
blebbistatin, which serve to decouple excitation from contraction,
can be missed by electrophysiological readouts but can be detected
by the impedance-based measurement of cardiomyocyte beating. These
observations demonstrate that impedance-based assay system can
detect modulation of ion-channel and non-ion channel targets
affecting both excitation and contraction of cardiomyocytes.
[0110] Also demonstrated is that a number of drugs that have been
shown to induce ventricular arrhythmia in the clinic and
subsequently withdrawn from the market display reproducible
impedance-based signature beating profiles in a time and
dose-dependent manner (FIGS. 13A-B and FIG. 15). These signature
beating profiles are qualitatively similar to early after
depolarization (EAD) phenomenon that is observed by manual patch
clamp recording of cardiomyocytes after exposure to compounds and
drugs that inhibit ERG channel activity and consequently I.sub.kr
current (Fermini et al., 2003). While the appearance and mechanism
of EADs are diverse, it is generally agreed that calcium
homeostasis which can influence contraction plays an important role
in various forms of EADs (Volders et al., 2000). It is thought that
drug-induced block in I.sub.kr current can delay repolarization
leading to delayed inactivation of calcium channels and
consequently, the resulting late inflow of calcium contributes to
emergence of EADs (Fermini et al., 2003). It is also important to
mention that manifestation of arrhythmia by mechanisms other than
ERG block and QT prolongation cannot be discounted. For example, it
has been shown that certain compounds which shorten both APD and QT
interval can also cause arrhythmia (Lu et al., 2008). While it
remains to be shown whether such drugs can also induce
characteristic EAD-like profiles in the assay system described
here, it underscores the need for implementation of an assay system
which can assess the integrated response of ion channel and non-ion
channel targets to capture the full range of drug responses.
[0111] While the cardiotoxic side effect of most drugs is primarily
viewed as a function of the concentration of the drug and its
possible adverse interaction with other compounds, it must be
emphasized that the time-dependent response of cardiomyocyte to a
given concentration exposure is also an important parameter. This
point is especially relevant for cardiac function where
time-dependent heart rate variability or dynamics of periodicity
could be a key determinant of proarrhythmic potential (Bass et al.,
2008). Furthermore, an increasing number of drugs are found to
inhibit hERG function by dual mechanisms of short-term channel
block and long term hERG trafficking defects that operate over
different time and concentration scales (van der Heyden et al.,
2008). The non-invasive nature of the impedance readout allows for
monitoring of cardiomyocyte response from millisecond time frame to
minutes, hours and days in real-time and therefore well suited to
capture time series data for these complex responses. The utility
of this feature was demonstrated with respect to treatment of mouse
embryoninc stem cell derived cardiomyocytes (mESCC) with both
pentmidine and doxorubicin (FIGS. 14A-C). Pentmidine, a hERG
trafficking inhibitor associated with TdP in the clinic had a major
impact on beating rate and beat duration that was elicited about
900 min after initial exposure (Kuryshev et al., 2005). Standard
manual patch clamp techniques or planar patch clamp techniques,
while the golden standard, and most likely would have missed this
effect because action potential recordings are typically performed
within the first hour after compound exposure. Doxorubicin, an
anthracycline affected both the beating rate and periodicity of
beating while causing progressive and dose-dependent decrease in
overall impedance signal of the cells (within 24 hours after
compound exposure) most likely due to loss of viability (FIGS. 14A
and B). Therefore, the ability to simultaneously capture the
dynamicity of beating rate changes coupled with overall changes in
impedance to reflect the global compound effect on cell health
within the same assay can provide additional information regarding
mechanism of toxicity.
EXAMPLES
Example 1: Detection of Changes in Cardiomyoctye Beating in
Response to Administration of Pharmacologically Active Agents
[0112] Cell culture. Mouse ES cell-derived cardiomyocytes (Cor.At)
were obtained from Axiogenesis (Cologne, Germany, catalogue number
XCAC-1010E, Lonza Cologne). The cells were kept in liquid nitrogen
until thawed and cultured according to protocol provided by
Axiogenesis with slight modifications. Briefly, each well of the
E-PLATE (ACEA Biosciences Inc., San Diego, Calif.) was coated with
50 .mu.l of a 1:100 diluted fibronectin (FN) solution (F1114,
Sigma-Aldrich, USA) and incubated at 4.degree. C. over night.
Subsequent to removal of FN, the wells were washed with PBS and
followed by cell seeding. The cells were thawed at 37.degree. C. in
a waterbath, transferred to 15 mL conical tube containing 9 ml
fresh Cor.At complete culture medium (XCAM-250E, Lonza Cologne,
Germany), centrifuged at 100 g for 5 minutes and the medium was
replaced with small volume of fresh Cor.At complete culture medium
containing puromyocin at final concentration of 10 .mu.g/ml. The
cells were counted and the percentage of viable cells determined by
trypan blue exclusion method.
[0113] Monitoring of cardiomyoctye attachment and contraction.
About 40000-60000 viable cells were seeded per well of a 96 well
E-PLATE (ACEA Biosciences Inc., San Diego, Calif.) and the cells
were monitored using the xCELLigence RTCA Cardio system (Roche
Applied Science and ACEA Biosciences). Cell culture medium was
replaced once on a daily basis. Typically, drug treatment was
initiated 60-80 hours after cell seeding depending on seeding
density. Data collection is controlled by software program which
operates the hardware and allows the user to define the sampling
frequency and sampling window. Sampling frequency is defined as the
number of times during an experimental run the beating is sampled
and the sampling window is defined as the duration of time that the
beating is actually measured. For example if the sampling frequency
is 15 minutes and sampling window is for 5 second means that each
15 minute the system will record beating data for 5 seconds. In a
typical experiment, prior to compound treatment the sampling
frequency is once every hour and the sampling window is 20 seconds.
5 min prior to treatment, the cells are sampled every minute for 20
seconds to establish baseline recording. After treatment, the
sampling frequency is every minute for the first hour, every 5
minutes for the second hour and every 15 minutes for 3-24 hours.
The sampling window for each recording is fixed at 20 seconds.
After the data acquisition, the RTCA Cardio software is used to
calculate the parameters such as beating rate, amplitude, beating
period, normalized beating rate, normalized amplitude, and beating
rate irregularity (BRI) index and perform subsequent basic
statistics, like average and standard deviation and further supply
EC50 values for dose-response testing.
[0114] Terms and Analysis Parameters. Each measured beating cycle
corresponds to the excitation-contraction coupling of the
cardiomyocytes. The typical measured beating pattern is illustrated
in FIG. 7 and FIG. 8. The beatings are composed of a sequence of
positive peaks (+P) and negative peaks (-P). The Cell Index
difference between one negative peak to the following positive peak
is defined as amplitude. The time between each positive peak is
defined as beating period and the beating rate is calculated based
on each beating period to derive how many beatings occurred in one
minute. Three time-related parameters, rise time T.sub.r, falling
time (T.sub.f, or termed as decay time T.sub.d), and half-amplitude
width T.sub.IBD50, resolve the temporal beating characteristic.
[0115] For data analysis, the related parameters are calculated for
every beating within one recording period and the average and
standard deviation are derived correspondingly. In order to compare
the effect of tested compounds, beating rate or amplitude after
compound treatment are normalized to the same time point before
compound treatment to obtain the normalized beating rate or
normalized amplitude. In order to evaluate the degree of
arrhythmia, the beating rhythm irregularity (BRI) index is derived
based on the coefficient of variation (i.e. standard deviation
divided by average) of the beating period during one record
period.
[0116] Multi-Electrode Array. For the culture of the mouse ES
cell-derived cardiomyocytes a sterilized substrate-integrated
planar standard MEA (59 TiN electrodes and a grounded reference
electrode, 8.times.8 electrode grid, electrode spacing 200,
electrode diameter 30, glass ring (Multi Channel Systems GmbH,
Reutlichen, Germany), 10 .mu.l of the 1:100 diluted fibronectin
solution (F1141, Sigma-Aldrich) was placed exactly on the
microelectrode area of the MEA and incubated for at least 3 hours
at 37.degree. C. in an humidified incubator. Afterwards residual
coating solution of taken off and 20 .mu.l with 2.times.10.sup.4
cardiomyocytes were placed on the coated electrode area and the
complete MEA was incubated for another 3 hours at 37.degree. C. in
the incubator to establish cell adhesion before 1 ml of Cor.At
culture medium was applied. The MEA was connected to the amplifier
and data-acquisition system (Multi Channel Systems) with band pass
filter characteristics of 0.5 Hz to 1 kHz. Spontaneous electrical
activity was recorded with software (MC Rack; Multi Channel
Systems) (Stett 2003). Data were recorded simultaneously from 59
channels with a sampling frequency of 10 kHz.
[0117] Cardiomyocytes on MEAs were kept in an incubator at
37.degree. C. during the whole time period of the assay. The cells
were equilibrated to the assay buffer (IMDM+0.1% FCS) for at least
45 min prior to baseline recording and subsequent substance
application. After that, three increasing concentrations of the
test compound were applied consecutively for 15 min each.
Additional wash-out period with was at least 45 min. Analyzed
parameters from extracellular recordings did not alter in a time
dependent manner in time-matched control experiments of the vehicle
(water or 0.1% DMSO) during all experimental phases.
[0118] Raw data from electrode array recordings was analyzed
offline. Frequency was determined as the reciprocal value of the
inter spike intervals of the field action potentials and field
action potential duration was calculated according to Halbach et
al. (2003) (Halbach et al., 2003). Frequency correction of the
field potential duration was assessed according to Mitchell et al.
(Mitchell et al., 1998).
[0119] Data are presented as mean values.+-.standard error of the
mean in percent of baseline. In order to evaluate compound-induced
effects relative to control measurements, differences between the
control group and the compound measurements were tested for
statistical significance by means of unpaired Student's t-test.
[0120] Microelectronic monitoring of cardiomyocyte beating. To
characterize the beating, mESCC were seeded in the wells of the
E-Plate at a density of 40,000 cells/well. The cells were monitored
up to 96 hours in culture (FIG. 9A) and the beating activity was
recorded at 12, 24, 48, 72 and 96 hours (FIG. 9A, arrows) for a
total duration of 20 seconds (FIG. 9B). Interestingly, within 24
hours after seeding the cells no consistent beating activity could
be detected even though, under the microscope clusters of
asynchronously beating cardiomyocytes, which have not had an
opportunity to form a syncytium could be detected (data not shown).
However, within 48 hours the individual clusters begin to form
clear connections and the entire monolayer of cardiac cells in the
bottom of the well begin to beat in a synchronous manner. Likewise,
based on impedance recording, reproducible beating activity is
detected by 48 hours (FIG. 9B). The beating rate at 48 hours is
approximately 80 beats/minute and progressively increases with time
reaching almost 250 beats/minute after a month in culture. These
observations are consistent with electrophysiological monitoring of
action potential duration in mouse ES cell-derived cardiomyocytes
(Fijnvandraat et al., 2003).
[0121] In order to analyze the curves and quantify beating
activity, 3 different analysis parameters were derived;
T.sub.IBD50, T.sub.r and T.sub.d. T.sub.IBD50 is a parameter which
measures the duration (ms) between the rise and fall of beat cycle
at 50% of maximal amplitude. T.sub.IBD50 values for mESCC at
corresponding times are shown in FIG. 9B. At 48 hours the
T.sub.IBD50 value is 142.+-.4.6 ms which decreases to 105.+-.2.4 ms
by 96 hours. The initial rise in amplitude denoted as T.sub.r is
relatively fast and depending on the time of recording can vary
from 29.+-.5.1 ms to 38.+-.1.4 ms (FIG. 9B). The decay time (or the
falling time as shown in FIG. 8), denoted as T.sub.d, which
reflects the time the signal decays from 80% of peak height to 20%
of peak height is longer compared to T.sub.r and can range from
88.+-.7.2 ms to 124.+-.12.0 ms, depending on the time of recording
(FIG. 9B). Interestingly, the kinetics of rise and fall of
impedance mirrors that of calcium in mouse embryonic cardiomyocytes
(Rapila et al., 2008) and it is possible that T.sub.r and T.sub.d
may reflect the time for two alternating phases of the beating
cycle, namely contraction and relaxation.
[0122] To determine if the impedance signal is reflective of
physical contraction and relaxation cycle of mESCC, we used an
inhibitor of the myosin heavy chain ATPase activity, blebbistatin,
shown to inhibit cardiomyocyte contraction (Kovacs et al., 2004).
As shown in FIG. 9C, blebbistatin treatment of mESCC resulted in
significant inhibition of impedance signal, which was restored
after washing the wells and culturing the cells in media without
blebbistatin. Interestingly, at concentration which blebbistatin
inhibited impedance measurement of beating activity, no effect on
action potential duration was detected using field potential
recording (FIG. 9D). The inability of electrophysiological readout
to detect blebbistatin effect has also been confirmed using
isolated rat and mouse ventricular myocytes, isolated rabbit
ventricle, and Langendorff-perfused rabbit hearts (Dou et al.,
2007; Fedorov et al., 2007) indicating that electrophysiological
readouts may miss the potential side effect of compounds depending
on whether the compounds affect the electrical or mechanical
aspects of cardiomyocyte contraction. Overall, the results
presented thus far demonstrate that impedance readout can be used
to monitor the rhythmic contraction/relaxation cycle of mESCC in
culture over a prolonged duration and in combination with
electrophysiological readouts may be able to detect compounds that
decouple excitation and contraction.
[0123] Pharmacological assessment of mESCC using impedance
monitoring. Using specific pharmacological modulators of ion
channel and non-ion channel targets, we set out to dissect specific
events of the excitation/contraction cycle in mESCC. First, the
time and dose-dependent effect of various ion channel modulators of
calcium, sodium and potassium channels were tested (FIGS. 10A-C and
FIGS. 11A-C). For these experiments mESCC were thawed, seeded in
the wells of the E-Plate, cultured for 3 days, treated with
increasing concentrations of the compounds and monitored for 24
hours using the RTCA Cardio system.
[0124] Assessment of voltage-gated calcium channels. Embryonic stem
cell-derived cardiomyocytes are known to undergo spontaneous
contractions due to intracellular calcium oscillations mainly
initiated from the sarcoplasmic reticulum (SR) (Sachinidis et al.,
2003). It is also believed that during SR-driven spontaneous
activity, the plasmalemmal voltage activated calcium influx could
provide a compensatory mechanism for restoring depleted calcium
pools in the SR (Rapila et al., 2008). Application of isradipine, a
well known voltage activated L-type calcium channel blocker of the
dihydropyridine class (Triggle, 2003) caused a progressive time and
dose-dependent decrease and inhibition of beating activity,
indicating that calcium entry through L-type calcium channels is
required for beating (FIG. 10A). The half maximal dose-response
value for isradapine induced inhibition of beating activity based
on measurement of normalized beating rate and amplitude is 19.7 nM
and 42.3 nM respectively (at 5 min time point after compound
addition; FIG. 12). These values are consistent with efficacy of
isradipine tested in isolated rabbit heart (Mellemkjaer et al.,
1992) as well as recombinant HEK-293 cells stably expressing the
human Cav1.2 (Balasubramanian et al., 2009) (FIG. 15). The compound
(S)-(-)Bay K 8644 is also of the dihydropyridine class, but acts in
an agonistic mode to activate voltage-gated calcium channels
(Franckowiak et al., 1985; Schramm et al., 1985). Treatment of
mESCC with (S)-(-)Bay K 8644 resulted in a dose and time dependent
effect that substantially increased the beating rate which
persisted for up to 12 hours at higher concentration and declined
by 24 hours (FIG. 10B). The increase in beating rate is consistent
with the reported ionotropic action of this compound and the EC50s
obtained (77 nM) for beating rate is consistent with previously
published reports using rat ventricular myocytes (Zahradnikova et
al., 2007) (33 nM; data not shown).
[0125] Assessment of potassium channel modulators. Next, the effect
of Chromanol 293B, an inhibitor of slow activating delayed
rectifier K.sup.+ current (I.sub.ks) (Bosch et al., 1998; Fujisawa
et al., 2000; Ono et al., 2000) was tested (FIG. 10C). While at the
highest dose (100 .mu.M) Chromanol 293B treatment resulted in
complete inhibition of cardiomyocyte beating activity, at
intermediate doses it slows down the beating rate (69% and 80% of
control at 25 .mu.M and 3.13 .mu.M, respectively 5 min post
compound addition) and also prolongs the beat duration (13.0 ms and
21.1 ms at 25 .mu.M and 3 .mu.M respectively and at 5 min post
compound addition). The I.sub.ks is mainly involved in the
repolarization phase of the action potential and its inhibition by
Chromanol 293B leads to increased action potential duration (APD)
of canine ventricle myocytes (Volders et al., 2003) and stem
cell-derived human cardiomyocytes (Peng et al.) as measured by
electrophysiological techniques. The increased APD has been shown
to slow down the decline of calcium concentrations and thereby may
prolong the contraction phase of cardiomyocytes (Bouchard et al.,
1995).
[0126] The rapid activating component of the delayed rectifier
current (kr) is also involved in the repolarization phase of
cardiac action potential and is mainly mediated through the ERG
channel (Brown, 2005). The effect of E4031, a potent ERG channel
inhibitor, was also tested using mESSC in a time and dose-dependent
manner (FIG. 11A). As shown, E4031 treatment interrupted that
normal rhythmicity of beating, especially at high concentrations
(200 nM-800 nM) and resulted in prolonged beat durations which are
accompanied by plateau oscillations. This phenomenon was typical of
other ERG blockers as well (see next section). At the doses tested
the cells appear to recover from the effect of E4031 by 24 hours
after treatment. Based on normalized beating rate and beat rate
irregularity parameter, the half maximal value obtained is 27 nM
and 57 nM, respectively and is consistent with the reported
IC.sub.50 for E4031(10 nM) using stem cell-derived human
cardiomyocytes with patch clamp technique (Peng et al.).
[0127] Assessment of sodium channel modulators. Voltage-gated
Na.sup.+ channels are primarily responsible for the Na.sup.+
current and the depolarization phase of cardiac action potential.
Based on gene expression and electrophysiological data, the Scn5a
gene product, which encodes for the .alpha.-subunit of
voltage-gated Na.sup.+ channel, is present and functional within
mESCC. Treatment of mESCC with Tetrodotoxin (TTX), a potent and
selective inhibitor of voltage-gated Na.sup.+ channels (Narahashi,
2008), led to a dose-dependent decrease in beating rate of mESCC
which is sustained at the higher concentrations for the entire
duration of 24 hours (FIG. 11B). The apparent half-maximal
dose-response value obtained for inhibition of mESCC beating is
about 0.28 .mu.M (FIG. 12).
[0128] Assessment of chronotropic agents. Activation of the
sympathetic nervous system and neuro-hormonal regulation through
the .beta.-adrenergic receptor is a major mechanism controlling
rate and contractility of the cardiac tissue (Bers, 2002). The
protein machinery responding to .beta.-adrenergic receptor
stimulation is present and functional within mESCC and its agonists
are well characterized chronotropic and ionotropic stimulants
(Maltsev et al., 1999). Therefore we sought to test whether
.beta.-adrenergic receptor stimulation could be detected by the
RTCA Cardio system. Treatment of mESCC with Isoproterenol, a
.beta.-adrenergic receptor agonist, increased the contraction
frequency of mESCC in a dose and time-dependent manner while
decreasing the overall duration of each beat (FIG. 11C). The
overall effect is similar to the L-type calcium channel agonist
(S)-(-)Bay K 8644 (FIG. 10B) and is consistent with the observation
that stimulation of .beta.-adrenergic receptors leads to activation
of L-Type calcium channels (Maltsev et al., 1999). The data
presented in this section demonstrates that impedance is a
sensitive readout to assess cardioymocyte beating and its
modulation by pharmacologically active compounds which alter
cardiac electrophysiological and/or contractile properties.
Compound treatment produces both time and concentration-dependent
responses which are captured in real-time by the RTCA Cardio
system. Both acute and long term assessment of beating activity may
provide additional mechanistic insight as demonstrated for ERG
channel inhibitors in the next section.
Example 2: Mechanism-Dependent Compound Toxicity Screening:
Identification of Impedance-Based Pro-Arrhythmic Signatures
[0129] To test the utility of RTCA Cardio system for pre-clinical
cardio-safety screening two complementary approaches were
undertaken. First, 4 drugs withdrawn from the market due to
increased incidence of TdP (Fermin et al., 2003) were screened in a
dose-response manner using mESCC (FIG. 13A). These compounds have
subsequently been shown to also inhibit hERG channel activity
(Brown, 2005). All four compounds significantly affected beating
rate in a dose-dependent manner (FIG. 13A) and produced beating
irregularities that were consistent with those observed for E4031
in terms of beating waveform, suggesting a common underlying
mechanism (FIG. 13B). We speculate that the plateau oscillation
phenomenon observed by hERG channel blockers maybe related to early
after depolarization (EAD) effect caused by blocking of the hERG
current leading to premature activation of voltage-dependent L-Type
calcium channels, calcium entry and culminating in premature
contractions (Rubart et al., 2005). These signature beating
waveforms were also observed for other drugs that are known to
interact with and block ERG activity (FIG. 15 and see below). In
order to better quantify the beating irregularities we derived a
kinetic parameter referred to as beating rhythm irregularity (BRI)
index which represents the coefficient of variation (standard
deviation divided by average) of beating rate periods. Based on
this parameter we derived half maximal concentrations for E4031
(FIG. 12), astemizole, cisapride, droperide and sertindole (FIG.
13A) which are 57 nM, 290 nM, 2700 nM, 2100 nM and 290 nM,
respectively. The respective values obtained here are within the
range reported for these compounds using electrophysiological
methods (data not shown). These findings suggest that
impedance-based beating profiles could be used in a predictive
manner to screen for and identify compounds that may have
off-target interactions with ERG channel.
[0130] Next, a compound library containing 50 proarrhythmic and
antiarrhythmic compounds was also screened at 3 doses (10 .mu.M, 1
.mu.M and 0.1 .mu.M) (FIG. 15). As shown, all known hERG blockers
with the exception of Terfenadine displayed beating profiles
consistent with those shown in FIG. 13B. A more extensive
dose-response profiling of Terfenadine may be required in order to
observe the signature beating profile similar to other hERG channel
inhibitors. In addition, in this screen, compounds modulating other
ion channel targets such as calcium and sodium also profoundly and
dose-dependently affected beating activity.
Example 3: Assessment of Short and Long Term Cardiac Liability
Using Impedance-Based Systems
[0131] The true test of any in vitro assay utilized in preclinical
safety assessment depends on its ability to model and predict in
vivo effect in the clinic. Thus far we have shown compounds
modulating ion channel activities in cardiomyocytes can be detected
by the RTCA Cardio system. However, there are a number of drugs
whose cardiac liability in the clinic extends beyond its propensity
to just cause arrhythmia; for example the chemotherapeutic agent,
doxorubicin, has been shown to induce arrhythmia (Singal et al.,
1998) as well as cardiotoxicity by interfering with mitochondrial
function (Minotti et al., 2004). Therefore, we wanted to determine
if the RTCA Cardio system in combination with mESCC can model and
predict the complex effects of doxorubicin. As shown in FIG. 14A,
treatment of mESCC with doxorubicin results in time and
dose-dependent decrease in global impedance readout, presumably due
to loss of cardiomyocyte viability. Likewise, FIG. 14B, shows the
dose- and time-dependent effect of doxorubicin on cardiomyocyte
beating within the same assay. Doxorubicin treatment of mESCC leads
to significant decrease in overall beating rate and also induces
irregular beating pattern that has features of compounds that
induce arrhythmia. While it may be difficult to draw direct
parallels between in vitro assays and clinical observations, both
acute and chronic effect of doxorubicin resulting in arrhythmia has
been documented in the clinic (Minotti et al., 2004).
[0132] The mode of interaction of drugs with various targets within
cardiomyocytes may be direct as shown for the various ERG channel
blockers and those of sodium and calcium channels or it could also
be indirect affecting such processes as the folding or transport of
ion channel proteins to the membrane surface of cardiomyocytes
(Dennis et al., 2007) and therefore may go undetected in most
conventional safety studies which are geared towards identification
of direct ERG blockers. This point is best exemplified by the
compound pentamidine, which in the U.S. is used as a second line of
treatment of Pneumocystis carinii pneumonia, a common opportunistic
infection in patients with impaired immune function. Pentamidine
has been shown to affect the transport of the ERG channel to the
membrane in heterologuous expression systems as well as in cardiac
myocytes with repolarization being delayed as a direct consequence
(Dennis et al., 2007; Kuryshev et al., 2005). Since this compound
affects ERG channel activity indirectly, its effect will be
manifested in a time-dependent manner and difficult to capture by
standard patch clamp techniques which are limited to the first hour
of recording time. We tested the effect of pentamidine on mESCC in
a time dependent manner (FIG. 14C). Administration of pentamidine
at a final concentration of 20 .mu.M has no noticeable effect on
beating rate and amplitude well into 900 min after compound
addition, at which point the beating rate slows down and the
beating duration is significantly delayed, most likely due to
extended repolarization phase. These observations highlight the
importance of monitoring compound effect in a time-dependent manner
in order to resolve the effect of compounds on both early and
longer term function of cardiomyocytes and to obtain greater
mechanistic understanding.
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