U.S. patent application number 13/932033 was filed with the patent office on 2014-03-06 for cardiomyocytes-containing device and method for manufacturing and using the same.
The applicant listed for this patent is Koninklijke Philips N.V.. Invention is credited to Ronald DEKKER, Anja VAN DE STOLPE.
Application Number | 20140065657 13/932033 |
Document ID | / |
Family ID | 40561887 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140065657 |
Kind Code |
A1 |
DEKKER; Ronald ; et
al. |
March 6, 2014 |
CARDIOMYOCYTES-CONTAINING DEVICE AND METHOD FOR MANUFACTURING AND
USING THE SAME
Abstract
A method for developing a disease model for a disease that is
caused by or modified by stretching of cells, in particular a
cardiac disease model uses a device for determining the
cardiotoxicity of a chemical compound, comprising a substrate (10)
carrying a deformable stack (34), said stack being partially
detached from the substrate by a cavity (32) allowing an
out-of-plane deformation of the stack, said stack comprising a
first deformable layer (16), a second deformable layer (20) and a
multi-electrode structure (18) sandwiched between the first and
second deformable layers, the second deformable layer carrying a
pattern of cardiomyocytes (28) adhered thereto; and a liquid
container (26) mounted on the substrate for exposing the
cardiomyocytes to the chemical compound. A method of manufacturing
such a device is also disclosed.
Inventors: |
DEKKER; Ronald;
(VALKENSWAARD, NL) ; VAN DE STOLPE; Anja; (VUGHT,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Koninklijke Philips N.V. |
Eindhoven |
|
NL |
|
|
Family ID: |
40561887 |
Appl. No.: |
13/932033 |
Filed: |
July 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13147607 |
Dec 8, 2011 |
8506793 |
|
|
PCT/IB2010/005053 |
Feb 9, 2010 |
|
|
|
13932033 |
|
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Current U.S.
Class: |
435/29 |
Current CPC
Class: |
C12N 5/0657 20130101;
C12N 2535/10 20130101; G01N 1/30 20130101; G01N 33/5061 20130101;
C12N 2503/02 20130101 |
Class at
Publication: |
435/29 |
International
Class: |
G01N 1/30 20060101
G01N001/30 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 2009 |
EP |
09152378.7 |
Claims
1. A method for developing a disease model for a disease that is
caused by or modified by stretching of cells, the method comprising
the steps of: Attaching at least one cell to an adhesive surface
pattern, Stretching the at least one cell by an externally applied
force, and Measuring an action potential of the at least one cell
electrically, the action potential being monitored and/or
interpreted over time.
2. A method according to claim 1, wherein the stretching of the at
least one cell is in-plane and/or out-of plane with respect to the
adhesive surface pattern.
3. A method according to claim 1, wherein the percentage of cell
stretching in-plane is larger than 30% and the stretching time is
varied.
4. A method according to claim 1, wherein during the measurement
chemical or biological compounds are added.
5. A method according to claim 1, wherein the disease model is a
cardiac disease model.
6. A method for developing a disease model for a disease that is
caused by or modified by stretching of cells, the method comprising
the steps of: Attaching at least one cell to an adhesive surface
pattern, Stretching the at least one cell by an externally applied
force, and Measuring an action potential of the at least one cell
optically, the action potential being monitored and/or interpreted
over time.
Description
[0001] This application is a divisional of application Ser. No.
13/147,607, filed Dec. 8, 2011, which entered the U.S. national
stage from PCT/IB2010/05053, filed Feb. 5, 2010 and claims the
benefit of EP Application No. 09152378.7, filed Feb. 9, 2009.
FIELD OF THE INVENTION
[0002] The present invention relates to a device for determining
cardiotoxicity of a chemical compound.
[0003] The present invention further relates to a method of
manufacturing such a device.
[0004] The present invention yet further relates to a method of
determining the cardiotoxicity of a chemical compound using such a
device.
[0005] The present invention further relates to the use of the
device for drug target discovery and/or drug development.
[0006] The present invention further relates to a method for
developing a disease model for a disease that is caused by or
modified by stretching of cells, in particular a cardiac disease
model.
BACKGROUND OF THE INVENTION
[0007] Many drugs have cardiotoxic side effects, e.g. arrhythmias
or negative effects on the contractive capacity of the heart
muscle. Over the last years it has become evident that a common
side-effect of a number of drugs is a prolonging effect on the QT
interval in the cardiac cycle, which is an important cause of
drug-induced life threatening arrhythmias. For instance, during the
past years, the development of several drugs has been aborted in
late phases of preclinical testing or clinical trials, and even
post-marketing due to undesirable effects on the QT interval of the
surface electrocardiogram (ECG). A prolongation of this interval to
more than 440 to 460 msec may allow life threatening arrhythmias,
e.g. torsade de pointes (TdP), to occur and has been associated
with a wide variety of drugs.
[0008] This was acknowledged in 1998 when the Food and Drug
Administration (FDA) defined prolongation of the QT interval as a
major drug safety issue. Subsequently, identification of QT
prolongation and clinical torsade de pointes has led to the removal
of several drugs from the market in the United States, including
terfenadine, astemizole, thioridazine, and grepafloxacin, while
many others have been required by the FDA to carry additional
safety labeling warning of the potential risk. Currently, assessing
risk for delayed ventricular repolarization and QT interval
prolongation is part of the standard non-clinical evaluation of
NCE's as adopted by the FDA and EMEA for all drugs in
development.
[0009] Unfortunately, currently available preclinical in-vitro
cell-based model systems to test for cardiotoxicity are inadequate
for detecting the majority of these side-effects, while predictive
in-vivo animal studies are very expensive, as well as ethically
challenged. In addition, cardiotoxicity results obtained from
animal studies cannot be easily extrapolated to humans.
[0010] The testing process is further complicated by the fact that
these cardiotoxic effects of drugs may only become apparent during
actual cardiac muscle stretching and contraction as occurs in vivo
in the beating heart, especially during (strenuous) physical
exercise; and in cardiac diseases, for example diseases associated
with cardiac overload, e.g. heart failure, and diseases
characterized by inflammation, like during influenza infections.
Currently no testing model systems exist that simulate a normal
beating heart, in either a physiological situation, i.e. a
stretch-contraction cycle, or a pathophysiological situation, such
as excessive stretch/contraction against increased pressure,
associated with cardiac failure. Moreover, different drugs can have
different negative effects on the heart function. Some human
cell-based model systems are available for cardiotoxicity testing.
These model systems typically may consist of cardiomyocytes, either
animal or human, and either primary cardiomyocytes or stem
cell-derived cardiomyocytes on standard multi-electrode arrays
(MEA), as disclosed in "Pluripotent stem cell lines" J. Yu and J.
A. Thomson, Genes Dev. 2008, 22, p. 1987-1997. However the
usefulness of these systems is constrained by the fact that these
are static model systems not taking into account the dynamics of
the beating heart.
[0011] In `An Electro-Tensile Bioreactor for 3-D Culturing of
Cardiomyocytes` by Zhonggang Feng et al. in IEEE Engineering in
Medicine and Biology Magazine, July/August 2005, pages 73-79, a
bioreactor is disclosed which allows for the in-plane stretching of
a cardiomyocyte-containing gel layer disposed on a stretchable
silicone plate to simulate the mechanical and electrical response
of the myocardium in vivo. A drawback of this device is that it is
quite complex and not particularly suitable for cardiotoxicity
testing due to the fact that the cardiomyocytes are embedded in a
gel.
SUMMARY OF THE INVENTION
[0012] The present invention seeks to provide an improved device
for determining the cardiotoxicity of a chemical compound.
[0013] The present invention further seeks to provide a method of
manufacturing such an improved device.
[0014] The present invention yet further seeks to provide a method
of determining the cardiotoxicity of a chemical compound using such
an improved device.
[0015] The present invention further seeks to provide a method of
developing (cardiac) disease models using such an improved
device.
[0016] According to a first aspect of the present invention, there
is provided a device for determining the cardiotoxicity of a
chemical compound, comprising a substrate carrying a deformable
stack, said stack being partially detached from the substrate by a
cavity allowing an out-of-plane deformation of the stack, said
stack comprising a first deformable layer, a second deformable
layer and a multi-electrode structure sandwiched between the first
and second deformable layers, the second deformable layer carrying
a pattern of cardiomyocytes adhered thereto and a liquid container
mounted on the substrate for exposing the cardiomyocytes to the
chemical compound.
[0017] The presence of the cavity ensures that at least the central
region of the first deformable layer, e.g. an elastomer layer, is
not attached to the substrate, such that this region can move
freely, e.g. as triggered by a contraction of the cardiomyocytes.
The provision of the cavity ensures that the cardiomyocyte
contraction induced movement can be facilitated in a relatively
simple manner, thereby reducing the cost of the device of the
present invention compared to the devices available in the art.
[0018] In an embodiment, one end of the deformable stack is
detached from the substrate to facilitate the out-of-plane
deformation, e.g. curling, upon contraction of the cardiomyocytes.
This has the advantage that the cardiomyocyte contraction can be
monitored electrically through the electrode arrangement as well as
optically, through the amount of curling. In addition, the curling
principle ensures that the intrinsic resistance of the stack to the
deformation forces of the contracting cardiomyocytes is low, such
that the contraction is not significantly hampered by this
resistance.
[0019] In a preferred embodiment, the substrate comprises the
cavity, the stack extending over the cavity, wherein opposite ends
of the stack are attached to the substrate, thereby facilitating
the out-of-plane deformation of the stack by an externally applied
force. This has the advantage that the device can also be used to
train immature cardiomyocyte cells by stretching the cells during
the out-of-plane deformation of the deformable stack, such that the
differentiation and maturation process of the live cardiomyocyte
cells is accelerated. In addition, contraction of the
cardiomyocytes is still facilitated due to the fact that the
stretchable nature of the stack allows for an in-plane
cardiomyocyte-induced stretching of the deformable layers, such
that both cardiomyocyte stretching and contraction during diastole
and systole respectively can be simulated with this device in a
quantitative manner at the appropriate stretch-contraction cycle
frequency. This may stimulate further differentiation and
maturation of stem cell-derived cardiomyocytes.
[0020] The device may comprise an inlet for filling the cavity with
a fluid. In an embodiment, the fluid is a gas (e.g. air), such that
the out-of-plane deformation of the stack may be controlled by
controlling the gas (e.g. air) pressure inside the cavity, thereby
simulating the beating of the heart. This has the advantage that no
contact with the stack is required to invoke the out-of-plane
deformation, thus reducing the risk of contamination or damage to
the cardiomyocytes. In an alternative embodiment, the fluid is a
liquid, and the stack comprises the inlet. The provision of the
inlet in the stack further facilitates the stretching of the stack
during a contraction cycle of the cardiomyocytes, thereby for
instance enabling the use of a linear electrode array. In this
embodiment, any fluid placed in the container will envelop both
surfaces of the stack such that the load of the fluid on the stack
will effectively be zero. In this case, the out-of-plane
deformation of the stack may be mechanically invoked. Out-of-plane
deformations of the stack of 100% and more can be obtained.
[0021] In an embodiment, the stack is corrugated to make it
deformable. The corrugations further reduce the intrinsic
resistance of the stack to the contraction forces of the
cardiomyocytes, thereby improving the cardiomyocyte contraction
behavior of the device.
[0022] In accordance with another aspect of the present invention,
a method of manufacturing a device of the present invention is
provided. This method comprises growing an oxide layer of the
substrate; depositing the first deformable layer over the oxide
layer; depositing and patterning a conductive layer over the first
deformable layer, thereby forming the multi-electrode structure,
depositing a second deformable layer over the first deformable
layer, patterning the second deformable layer to provide access to
the multi-electrode structure, depositing an adhesive pattern over
the patterned second deformable layer, adhering cardiomyocytes to
the adhesive pattern, adhering the liquid container to the second
deformable layer and forming the cavity underneath the first
deformable layer.
[0023] This method has the advantage that the device of the present
invention can be formed at low cost, thereby making the method
attractive for large scale production purposes.
[0024] The order in which the steps of this method are performed
may be altered without departing from the scope of the present
invention. For instance, the steps of depositing an adhesive
pattern over the patterned second deformable layer and adhering
cardiomyocytes to the adhesive pattern may be performed after
forming the cavity underneath the first deformable layer. This has
the advantage that the cardiomyocytes can be applied to the second
deformable layer immediately prior to use, thereby ensuring that
the cardiomyocytes are in a good condition during use.
[0025] In an embodiment, forming said cavity comprises depositing a
sacrificial layer over the oxide layer prior to the deposition of
the first deformable layer, said sacrificial layer defining the
cavity volume and removing the sacrificial layer after the
deposition of the second deformable layer. This has the advantage
that the stack may be formed over the sacrificial layer rather than
over the cavity, thereby simplifying the manufacturing process
because no complex steps are required to form the stack over a
void.
[0026] In an alternative embodiment, forming said cavity comprises
applying a mask on the backside of the substrate; patterning said
mask to define the cavity area; etching the backside of the
substrate to expose the first deformable layer; and removing the
patterned mask from the backside of the substrate. This has the
advantage that the stack may be formed over the substrate rather
than over the cavity, thereby simplifying the manufacturing process
as previously explained.
[0027] Advantageously, the method further comprises forming a
corrugated pattern in the substrate prior to forming the oxide
layer. This has the advantage that the deformability may be
achieved by the flexibility of the corrugations, which aids the
contraction cycle of the cardiomyocytes as previously
explained.
[0028] In an embodiment, said corrugated pattern is formed through
milling. In an alternative embodiment, forming said corrugated
pattern comprises depositing a silicon oxide layer over the
substrate; depositing a silicon nitride layer over the silicon
oxide layer; patterning the silicon oxide and silicon nitride
layer, thereby exposing selected parts of the substrate; exposing
said selected parts to a series of etching steps to form said
corrugated pattern; and removing the silicon oxide and silicon
nitride layer. This has the advantage that the corrugated pattern
may be formed using readily available semiconductor processing
techniques, thereby limiting the complexity and cost of the device
manufacture.
[0029] Alternatively, said corrugated pattern may be formed by a
LOCOS oxidation step followed by an etching step in which the LOCOS
oxide is removed. This also has the advantage that the corrugated
pattern may be formed using readily available semiconductor
processing techniques, thereby limiting the complexity and cost of
the device manufacture.
[0030] According to yet a further aspect of the present invention
there is provided a method of determining the cardiotoxicity of a
chemical compound, comprising providing the device according to an
embodiment of the present invention; filling the container with a
medium comprising the chemical compound to expose the
cardiomyocytes to said compound; and measuring the response of the
cardiomyocytes to said exposure.
[0031] The use of the device of the present invention in such a
method provides an improvement in the accuracy of the
cardiotoxicity determination of chemical compounds such as trial
drugs.
[0032] The present invention further relates to a method for
developing a disease model for a disease that is caused by or
modified by stretching of cells, the method comprising the steps
of:
[0033] Attaching at least one cell to an adhesive surface
pattern
[0034] Stretching the at least one cell by an externally applied
force
[0035] Measuring an action potential of the at least one cell
electrically and/or optically, the action potential being monitored
and/or interpreted over time.
The method is in particular suitable for establishing a cardiac
disease model (among other disease models where cell stretch and
measuring ion channel activity and electric potential is relevant).
It is a big advantage that heart cell toxicity can be measured
under conditions simulating increased heart load (stress) during
physical exercise or other conditions associated with increased
cardiac output. It is a further advantage that heart cell toxicity
can be determined under conditions of heart disease, like cardiac
failure, cardiomyopathy etc. The effect of a specific genetic
variable on cardiotoxicity can be taken into account in the
method.
[0036] In an advantageous method, the stretching of the at least
one cell may be in-plane and/or out-of plane with respect to the
adhesive surface pattern. The percentage of cell stretching
in-plane is larger than 30% and the stretching time is varied.
[0037] Preferably, during the measurement chemical or biological
compounds are added.
[0038] The use of the device according to the invention allows the
development of accurate heart disease models for drug discovery and
development, also on a personalized basis, taking human genetic
variables into account.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0039] Embodiments of the invention are described in more detail
and by way of non-limiting examples with reference to the
accompanying drawings, wherein
[0040] FIG. 1 (a)-(g) is a schematic depicting a method of
manufacturing a device for determining the cardiotoxicity of a
chemical compound in accordance with an embodiment of the present
invention;
[0041] FIG. 2 is a schematic depicting a top view of the device for
determining the cardiotoxicity of a chemical compound as obtained
by the method of FIG. 1;
[0042] FIG. 3 (a)-(c) is a schematic depicting a device for
determining the cardiotoxicity of a chemical compound in accordance
with another embodiment of the present invention;
[0043] FIG. 4 is a schematic depicting an example electrode
arrangement for a device for determining the cardiotoxicity of a
chemical compound according to the present invention;
[0044] FIG. 5 is a schematic depicting an alternative example
electrode arrangement for a device for determining the
cardiotoxicity of a chemical compound according to the present
invention;
[0045] FIG. 6 (a)-(j) is a schematic depicting a method of
manufacturing a device for determining the cardiotoxicity of a
chemical compound in accordance with an alternative embodiment of
the present invention;
[0046] FIG. 7 (a)-(j) is a schematic depicting a method of
manufacturing a device for determining the cardiotoxicity of a
chemical compound in accordance with yet another embodiment of the
present invention; and
[0047] FIGS. 8-10 are schematics depicting alternatives for a part
of the method of FIG. 7.
DETAILED DESCRIPTION OF THE DRAWINGS
[0048] It should be understood that the Figures are merely
schematic and are not drawn to scale. It should also be understood
that the same reference numerals are used throughout the Figures to
indicate the same or similar parts.
[0049] The device for determining the cardiotoxicity of a chemical
compound of the present invention is based on the following general
structural principle. An elastomer-based stack is mounted over a
cavity in a substrate. This cavity effectively detaches a part of
the elastomer-based stack from the substrate, such that the
elastomeric nature of this part is facilitated to move out of the
plane of the surface of the substrate. Several embodiments of such
a device are contemplated, as will be discussed in more detail
below.
[0050] In FIG. 1, a method of manufacturing a first embodiment of
the device of the present invention is depicted. In a first step
(a), a substrate 10, e.g. a silicon wafer, a glass substrate or any
other suitable substrate material is provided and a dielectric
layer 12 such as a silicon oxide layer is formed over a surface of
the substrate 10. The formation of a dielectric layer 12 on a
substrate 10 is well-known to the person skilled in the art and
will not be further explained for reasons of brevity only.
[0051] In a next step (b), a sacrificial material 14 is deposited
over the dielectric layer 12 and subsequently patterned. The
patterned sacrificial material 14 defines the cavity to be formed.
Any suitable sacrificial material, such as a soluble or
decomposable polymer may be used. A suitable non-limiting example
of such a polymer is poly-N-isopropylacrylamide (PIPAAm).
[0052] In step (c), an elastomer-based stack is formed over the
patterned sacrificial material 14 and the dielectric layer 12. This
stack comprises a first elastomer layer 16, a patterned metal layer
18 formed on the first elastomer layer 16 and a second elastomer
layer 20 covering the patterned conductive layer 18 such that the
conductive layer is sandwiched between the first elastomer layer 16
and the second elastomer layer 20.
[0053] Any suitable elastomer layer may be used. In an embodiment,
the first elastomer layer 16 is a polydimethylsiloxane (PDMS)
layer, which may be applied by means of spin-coating. Any suitable
conductive material, e.g. a metal, may be applied on this layer.
For instance, a TiN layer or a Ti/Au layer stack may be deposited
over the PDMS layer 16 and subsequently patterned. A second PDMS
layer 20 may be spin-coated over the patterned conductive layer 18.
The patterned conductive layer typically comprises electrodes, bond
pads and connections between the bond pads and electrodes. This
will be explained in more detail later.
[0054] In step (d), contacts 22 to the electrodes and bond pads in
the patterned conductive layer 18 are formed in any suitable
manner, e.g. by RIE etching. This is followed by the application of
a fixating layer 24 to an area of the second elastomer layer 20
over the electrodes in the patterned conductive layer 18 for
fixating the live cardiomyocyte cells to the elastomer-based stack,
as shown in step (e). The live cardiomyocyte cells may be any
suitable type of cardiomyocytes, such as human or animal primary
cells, or human stem cell-derived cells.
[0055] An example of a suitable material for the fixating layer 24
is fibronectin, which may be applied by stamp-, screen- or
ink-jetprinting. The cardiomyocytes may subsequently be bonded to
this layer. It will be obvious to the skilled person that
fibronectin is a non-limiting example of such a fixating layer, and
that other suitable adhesive materials may also be considered.
[0056] The size of the elastomer-based stack defining the electrode
array foil may be determined by (laser) cutting the stack and
sacrificial material layers, thereby providing a cut 25, which also
acts as an access to the sacrificial material 14. This step is
followed by step (f), in which a liquid container 25 is attached to
the device in any suitable manner, e.g. by gluing. Finally, the
sacrificial material is removed, as shown in step (f). For
instance, in case of the sacrificial material being PIPMAAm, this
may be removed by dissolving this layer in Tyrode's solution. This
has the advantage that the cardiomyocytes may already be present on
the fixating layer 24 due to the isotonic nature of Tyrode's
solution. In case of a thermally decomposable material, the removal
may be achieved by exposing the device to a temperature above the
decomposition temperature of the material. In this embodiment, care
has to be taken that a material is chosen that decomposes at a
temperature low enough to avoid damage to other layers of the
device. To this end, both the fixating layer 24 and cardiomyocytes
may be applied to the elastomer-based stack after removal of the
thermally decomposable material.
[0057] In an embodiment, the elastomer-based stack or foil has a
thickness in the range of 3-10 micron. In this range, the
elastomer-based stack or foil has a particularly good
flexibility.
[0058] FIG. 1(g) demonstrates the device according to this
embodiment of the invention in operation. The liquid container 26
is filled with a solution 30 comprising the chemical compound to be
tested on the cardiomyocytes 28. The solution 30 embedding the
cardiomyocytes may contain both the test compound in various
concentrations as well as varying concentrations of molecules, e.g.
electrolytes such as potassium, sodium, calcium, glucose, oxygen,
CO.sub.2, required to simulate different (patho)physiological
situations in vivo, such as conditions induced by strenuous
exercise.
[0059] The cavity 32 under the elastomer-based stack carrying the
cardiomyocytes 28 allows for the out-of-plane curling of this stack
as triggered by the contraction of the cardiomyocytes 28. The field
potential recorded by the electrodes in the patterned metal layer
18 during the stretch-contraction cycle of the cardiomyocytes 28
can be used to determine the effect of the chemical compound on the
conductivity in the ion channels of the cardiomyocytes 28.
[0060] In the context of the present invention, it should be
understood that the phrase `chemical compound` is not intended to
be limited to compounds intended for use as a pharmaceutical or to
single compounds only. In general, any substance, such as compound
mixtures, emulsions and solutions comprising one or more compounds
may be tested using the device of the present invention.
[0061] A top view of the electrode array of the device shown in
FIG. 1(f) prior to the removal of the sacrificial layer 14 is shown
in FIG. 2. The patterned conductive layer 18 comprises a plurality
of bond pads 110 that are connected to respective electrodes 114
through conductive connectors 112. The liquid container 26 is
shaped such that it envelopes all the electrodes 114. The cut 25 is
also shown, which defines the portion of the stack that can curl up
as previously explained. The cut 25 extends though the underlying
sacrificial layer 14. This facilitates the removal of this layer,
as previously explained. The fixating layer 24, e.g. the protein
fibronectin, is preferably patterned in the form of stripes (not
shown) in the length direction of the stack. This maximizes the
curling of the stack upon contraction of the cardiomyocytes 28
adhered to these stripes.
[0062] In an alternative embodiment of the present invention, the
cavity 32 is formed in the substrate 10, with the deformable stack
covering an access to the cavity 32 such that a change in pressure
on the elastomer-based stack will allow for a deformation of the
stack into or away from the cavity 32. In a first embodiment, an
elastomer-based stack may be used, the operating principle of which
is demonstrated in FIG. 3. As shown in FIG. 3(a), the substrate 10
comprises a cavity 32 extending through the substrate, which is
covered by the elastomer-based stack 34. The stack 34 may be formed
by the first elastomer layer 16, the patterned conductive layer 18
and the second elastomer layer 20 and typically carries a pattern
of cardiomyocytes 28 enveloped by the liquid container 26 as
previously explained. These features are not explicitly shown in
FIG. 3(a) for reasons of clarity only. The stack or foil 34 is
typically kept very thin to optimize its stretchability.
[0063] As shown in FIG. 3(b), the device may further comprise a
chamber 32' having an inlet 36. The chamber 32' is in communicative
contact with the cavity 32 such that the pressure in the cavity 32
may be regulated, e.g. reduced or increased by withdrawing or
adding a gas such as air via the inlet 36. This forces the stack 34
to stretch in a direction out of the plane of the substrate 10,
e.g. into the cavity 32 when reducing the pressure therein or away
from the cavity 32 when increasing the pressure therein.
Consequently, the live cardiomyocyte cells adhered to the stack 34
are also stretched in this process. In FIG. 3(c), the autonomous
contraction of the cardiomyocytes triggers an in-plane deformation
of the stack 34, which comprises a thickening (contraction) of the
stack 34 under the cardiomyocytes and a thinning (stretching) of
the stack 32 outside the area in which the cardiomyocytes are
located.
[0064] The fact that the device as demonstrated in FIG. 3(a)-(c)
has a stack 34 that can be stretched has two main advantages.
Firstly, repetitive stretching may be applied to immature stem
cells to differentiate these stem cells into cardiomyocytes,
thereby yielding a device for determining the cardiotoxicity of
chemical compounds that comprises fully differentiated
cardiomyocytes, which improves the relevance of the clinical data
obtained with this device. Secondly, the stack 34 may be stretched
in-sync with the contraction rhythm of the cardiomyocytes to
resemble the beating heart. This for instance allows for the
attached cardiomyocytes to be passively stretched to allow ion
channel measurements in a dynamic cardiomyocyte model system
mimicking the heart at rest and under controlled
(patho)-physiological stress. The cardiomyocyte contraction rhythm
may be autonomous or electrically induced.
[0065] The electrode arrangement in the stack 34 may take any
suitable shape. In an embodiment shown in FIG. 4, the electrode
arrangement comprises a circular arrangement covered by a
cylindrical liquid container 26 for holding a liquid containing
nutrition and/or one or more chemical compounds, i.e.
pharmaceutical drugs. The container 26 envelopes a radial pattern
of adhesive strips 24, e.g. fibronectin strips to which the
cardiomyocytes are fixed, e.g. adhered. The radial pattern of
stripes promotes the contraction of the cardiomyocytes in a radial
pattern, causing a thickening of the central portion of the stack
34, as previously explained. Four stripes are shown for reasons of
clarity only. The stack 34 may carry any suitable number of
stripes. The pattern of electrodes 114 is aligned with these
strips. The electrodes 114 are connected to bond pads 110 via
connectors 112, as previously explained. The bond pads 110 may be
arranged in any suitable pattern such as a rectangular or square
pattern.
[0066] At this point, it is emphasized that the stretchable nature
of the stack 34 does not have to be achieved by the use of
elastomeric materials. Alternatively, the stack 34 may be
corrugated, such that the necessary stretchability is achieved
through the corrugations, as will be explained in more detail
later. For such embodiments, materials other than elastomers may be
used. FIG. 5 shows an example electrode arrangement of such a
corrugated stack. The connections 112 from the bond pads 110 to the
electrodes 114 are of a substantially linear nature and run in a
direction perpendicular to the corrugations 140 in the stack. The
cardiomyocyte stripes typically also run in the direction
perpendicular to the corrugations 140 in the stack.
[0067] To facilitate the stretching of the stack arrangement of
FIG. 5, openings 120 are provided alongside the stack. As a
consequence, the liquids containing the nutrition and the chemical
compounds to be tested, e.g. pharmaceutical drugs, will envelope
both sides of the stack, i.e. also fill the cavity 32, resulting in
a "zero" load on the stack as a result of the weight of the liquid.
In this case, the out-of-plane stretching of the stack may be
invoked mechanically. It is pointed out that a stack comprising the
circular electrode arrangement of FIG. 4 may also comprise openings
120 to reduce the "zero" load on the stack.
[0068] In a preferred embodiment, the patterned conductive layer 18
in the elastomer-based stack comprises Ti/Au as the conductive
material because it has been shown that thin titanium and gold
layers can be stretched up to 100%, thus allowing the conductive
pattern to be stretched without damage. Alternatively, TiN or
another suitable stretchable conductor may be considered.
[0069] FIG. 6 schematically depicts a method of manufacturing a
device according to FIG. 3 in which the stack 34 is
elastomer-based. In step (a), a silicon substrate 10 having a
thickness of around 300-400 micron is supplied and its back side is
provided with a suitable hard-etch mask 50, e.g. LPCVD grown
silicon nitride (Si.sub.3N.sub.4). The etch mask 50 is patterned to
define the cavity to be formed in the substrate 10. In other words,
the etch mask pattern defines the position and size of the
stretchable area.
[0070] In next step (b), a dielectric layer 12, e.g. a thermal
oxide layer, is grown on the front side of the substrate 10 to a
thickness in the region of 1 micron or less, e.g. 0.5 micron. This
is followed by the deposition and patterning of a first elastomer
layer 16, e.g. a first layer of PDMS, as shown in step (c). This
layer may be deposited in any suitable manner, such as by
spin-coating. PDMS is a particularly suitable material because it
is bio-compatible and can be elongated up to 100%.
[0071] In step (d), a patterned conductive layer 18 is formed over
the first elastomer layer 16. This may be achieved in any suitable
way. For instance, a Ti/Au stack or TiN may be used as a conductive
material, and may be sputter-coated and patterned to form the
electrode and interconnection pattern, such as the pattern shown in
FIG. 4. Note that the first elastomer 16 has been patterned such
that the elastomer is absent underneath the bond pads to allow for
reliable wire-bonding.
[0072] The elastomer-based stack is completed in step (e), in which
a second elastomer layer 20, such as a second layer of PDMS is
deposited in any suitable manner to seal the interconnect patterns
112 and patterned to expose the electrodes 114 and bondpads 110
shown in e.g. FIG. 4.
[0073] Next, the cavity in the substrate 10 is formed, as shown in
step (f) by wet-etching the exposed back side of the substrate 10
with any suitable etchant, of which a HF/HNO.sub.3/acetic acid
(HNA) etchant is a non-limiting example (FIG. 6(f)). After removal
of the hard-etch mask in step (g), the substrate 10, which
typically is a wafer carrying multiple sensor devices, is diced in
step (h), thereby separating the individual sensor devices. In case
the dielectric layer 12 has not yet been fully removed by the
wet-etching step, a further wet etching step may be applied to
remove residual dielectric layer material. Finally, the container
26 is glued to the front side of the device in step (i). The
adhesive layer pattern for adhering the cardiomyocytes to the
elastomer-based stack, e.g. a fibronectin pattern, may be applied
inside the container 26, e.g. by stamping, after which the
cardiomyocytes are applied to this adhesive pattern. This is
preferably done immediately prior to use of the device to ensure
that the cardiomyocytes are `fresh` when being used.
[0074] FIG. 6(j) depicts an embodiment of the interconnections 112
between the bond pads 110 and the electrodes 114 of the conductive
layer 18. The interconnections 112 have been designed in a
meandering shape to further facilitate the stretching of the
elastomer-based stack.
[0075] FIG. 7 shows a first embodiment of a method to form a device
having a stack that can be deformed by virtue of the stack
comprising corrugations, as previously discussed in the description
of FIG. 6. In step (a), a silicon substrate 10 having a thickness
of around 300-400 micron is supplied and its back side is provided
with a suitable hard-etch mask 50, e.g. LPCVD grown silicon nitride
(Si.sub.3N.sub.4). The etch mask 50 is patterned to define the
cavity to be formed in the substrate 10. This step is substantially
identical to step (a) in FIG. 6. Next, as shown in step (b),
corrugations 10' are formed in substrate 10 in any suitable manner,
e.g. by milling or etching. It is noted that although the
corrugations are shown to have a triangular shape as a non-limiting
example only. Preferably, the corrugation profile has a more
rounded or wavy shape. How such a wavy corrugated pattern can be
achieved will be discussed in more detail later.
[0076] In step (c), a thermal oxide etch stop layer is grown,
followed by the deposition of a conformal first layer 16 in step
(d). The conformal material may be any suitable material. A
preferred candidate material is parylene because it is a
biocompatible material with an exceptional step-coverage that can
be CVD deposited at a room temperature. At the location of the bond
pads, openings are etched in the layer 16.
[0077] In step (e), the patterned conductive layer 18 is formed,
e.g. a TiN layer is sputter-coated and patterned, to form the
electrode and interconnection pattern. A second elastomeric layer
20, e.g. a second parylene layer is deposited to seal the
interconnections 112 and patterned to expose the electrodes 114 and
the bond pads 110, as shown in step (f).
[0078] In step (g), the cavity 32 is formed underneath the
stretchable area by wet-etching the exposed back side of the
substrate with HNA. An additional wet etching step may be applied
to remove any residual thermal oxide etch stop if necessary. After
removal of the hard-etch mask 50 in step (h), the individual
devices are separated by dicing the substrate wafer in step (i),
after which the container 26 is adhered to the front side of the
substrate 10 in step (j). The adhesive layer pattern for adhering
to the cardiomyocytes to the stretchable area, e.g. a fibronectin
pattern, may be applied inside the container 26, e.g. by stamping,
after which the cardiomyocytes are applied to this adhesive
pattern.
[0079] It is reiterated that it is preferable to provide the
substrate 10 with corrugations 10' having a rounded or wavy shape.
One of the reasons for this is that the subsequent layers to be
formed over the corrugations, such as the elastomer layers 16 and
20, can be more easily formed due to the fact that there is a
reduced risk of disruptions in the continuity of these layers,
which may occur in the sharp corners at the bottom of triangularly
shaped corrugations due to the difficulty of properly lining these
corners with such subsequent layers.
[0080] FIG. 8 shows a first embodiment of a method to form rounded
corrugations 10', which utilizes the etching behavior of silicon
when exposed to an isotropic wet etching step. In step (a), the
silicon substrate 10 is provided with a suitable patterned hard
mask 50', with the pattern reflecting the desired locations of the
recesses of the corrugations 10'. A suitable hard-etch mask such as
an LPCVD grown Si.sub.3N.sub.4 layer may be used.
[0081] In step (b), the exposed parts of the front side of the
substrate 10 are exposed to an isotropic wet etch mixture such as
HNA or a HF/HNO.sub.3/H.sub.2O mixture. The isotropic etching step
results in an under-etch which laterally extends to approximately
0.7 times the etch depth. Alternatively, a dry etch may be applied,
such as a CF.sub.4/O.sub.2 plasma. Finally, the hard etch-mask 50'
is removed in step (c). This yield a corrugation pattern in which
the `valleys` 10' in the substrate 10 are rounded, whereas the
`peaks` separating neighboring `valleys` are flat.
[0082] In a second embodiment, these `peaks` are also rounded, such
that a more wave-like, i.e. wavy corrugation pattern is achieved.
This is shown in FIG. 9. In step (a), an oxide layer 12' is grown
over the substrate 10 prior to the deposition of the hard-etch mask
50'. The oxide layer 12' and hard-etch mask 50' are patterned to
expose the regions of the substrate 10 in which the corrugation
`valleys` 10' are to be etched.
[0083] Next, as shown in step (b), the silicon substrate 10 is
isotropically etched to an intermediate depth, which is less than
the finally required depth. This etch does not affect the hard-etch
mask 50', e.g. Si.sub.3N.sub.4 mask 50', and only slightly affects
the oxide layer 12'. In a subsequent etching step (c), the oxide
12' underneath the hard-mask layer 50' is etched, for instance by
means buffered oxide etch consisting of a mixture of NH.sub.4F/HF.
It is noted that a certain amount of "over-etch" will be
required.
[0084] Next, the silicon is again isotropically etched in etching
step (d). Since the isotropic etch preferentially attacks sharp
silicon corners, this step will result in a wavy silicon pattern.
After removal of the hard mask 50' and residual oxide layer 12' in
step (e), the device manufacture can continue as outlined in FIG. 7
and its detailed description. The wavy pattern can be optimized by
adjusting the pitch between the patterns and the thickness of the
oxide layer 12'.
[0085] A "wavy" corrugated structure with a finer pitch may be
obtained by using LOCOS, as shown in FIG. 10. This will be briefly
described only because LOCOS techniques are well known to the
person skilled in the art. In step (a), a silicon substrate 10 is
provided carrying a suitable LOCOS stack consisting of a pad oxide
12' and an LPCVD deposited Si.sub.3N.sub.4 hard mask layer 50'.
This stack is patterned to expose the regions of the substrate 10
in which the corrugations 10' are to be formed. In step (b), the
exposed regions of the silicon substrate are thermally oxidized to
form the thermal oxide layer 52. The Si.sub.3N.sub.4 hard mask
layer 50' will not be oxidized; however at the edges of the
Si.sub.3N.sub.4 hard mask layer 50' the thermal oxide layer 52 will
extend underneath the nitride, i.e. form the well-known birds-beaks
associated with LOCOS oxidation.
[0086] The size and the shape of the birds-beak are determined by
the thicknesses of the pad-oxide layer 12', the Si.sub.3N.sub.4
hard mask layer 50' and the oxidizing conditions. By optimizing
these conditions and the pitch between the waves, a corrugated
structure will be obtained after removal of the Si.sub.3N.sub.4
hard mask layer 50' in step (c) and oxide removal in step (d).
[0087] Alternatively, the corrugated silicon pattern may be
obtained using gray-scale lithography or using resist flow.
[0088] It is should be mentioned that the fibronectin, or any other
suitable adhesive, can be applied by stamp printing, spray coating
in combination with photo-lithography. In a preferred embodiment,
the fibronectin is applied by ink jetprinting. The adhesive
fibronectin pattern preferably is applied after placement of the
container on the stretchable stack, after which the cardiomyocytes
are applied to this adhesive pattern.
[0089] It is further pointed out that the electrode array may be
supplemented with a plurality of sensors other than electrodes.
Non-limiting examples of such sensors include strain-gauges that
can measure the amount of force induced by the contraction of the
cardiomyocytes, and micro-calorimeters that can measure the amount
of heat produced by these cells.
[0090] Finally, it should be pointed out that although the
embodiments of the device of the present invention have been shown
to comprise passive devices only in the patterned conductive layer
18, the electrode array formed therein may alternatively contain
active devices for forming circuits that for example can perform
the function of signal amplification and signal shaping.
[0091] It should be appreciated that the device of the present
invention makes it possible to measure the cardiomyocyte ion
channel activity to detect QT elongation, i.e. the elongation of
the interval representing the duration of ventricular
depolarization and subsequent repolarization, measured from the
beginning of the QRS complex to the end of the T wave of the heart
rhythm, as well as other electrophysiological abnormalities in the
heart cell.
[0092] Compared to electrophysiological measurements performed in a
steady state system, where the effect of passive cardiomyocyte
stretch due to filling of the left ventricle during diastole are
not taken into account, the device of the present invention can be
used to develop a heart cell model which is capable of accurately
simulating a large variety of arrhythmias, such as arrhythmias due
to long QT syndrome that occur during physical exertion, or
pathophysiological conditions associated with increased cardiac
output (like fever, anemia), when both heart rate, end-diastolic
ventricular volume and filling pressure increase to induce the
required increase in cardiac output.
[0093] The direct relationship between the level of stretch of the
ventricular wall cardiomyocytes and the contraction force of the
cardiomyocytes is described in the Frank-Starling law. With
increased stretch, contraction force increases until a point is
reached where further stretch induces a reduction in cardiac
output. This has been described as electromechanical feedback. This
(patho)physiological stretching of the cardiomyocytes plays a role
in ion channel activity and proneness to arrhythmias. The
stretchable device of the present invention makes it possible to
measure ion channel activity under controlled (patho-)physiological
conditions of cardiomyocyte stretch and contraction.
[0094] Such measurements of ion channel activity as described above
can be performed in specific cardiac disease models, such as a
disease model for hypertrophic cardiomyopathy and heart failure.
Prolonged excessive stretching (associated with increased cardiac
load) of the cardiomyocytes in the cardiac ventricular wall leads
to hypertrophy, potentially resulting in heart failure. Longer
periods of controlled stretch-contraction cycles applied to the
cardiomyocytes which are adhered to the stretchable stack of the
device of the present invention, with excessive passive stretch
applied, are expected to induce cardiomyocyte hypertrophy and to
simulate heart failure, and continuous recording of ion channel
activity and contraction force may reveal information on the
underlying pathogenetic mechanism. The above-described model system
for hypertrophic cardiomyopathy can be used for drug target
discovery, i.e. the identification of specific drug target
molecules that play a causative role in the disease process and the
discovery of compounds that can be used to treat the disease, as
well as for drug development. Obviously, the cardiotoxicity of
chemical compounds may also be tested in these disease states.
[0095] The aforementioned disease models may be implemented by
exposure of the live cardiomyocyte cells to solutions that comprise
solutes present in the blood such as electrolytes, O.sub.2,
CO.sub.2, glucose and so on in concentrations that are indicative
of the simulated disease. Hence, the response of the cardiomyocytes
to such solutes can be used to gain a better understanding of the
effect of the simulated disease on these cells.
[0096] The method for developing a disease model for a disease that
is caused by or modified by stretching of cells, comprises the
steps of:
[0097] Attaching at least one cell to an adhesive surface pattern
(24)
[0098] Stretching the at least one cell by an externally applied
force
[0099] Measuring an action potential of the at least one cell
electrically and/or optically, the action potential being monitored
and/or interpreted over time.
[0100] To develop and build the disease model for drug target
discovery and drug development and drug toxicity measurement,
several experimental steps are performed:
1. The required viable cells, that represent the diseased tissue
are produced/cultured, according to existing protocols. They can be
obtained directly from cell lines, or from an animal or human
tissue source and prepared for culturing. They can also be obtained
from animal or human stem cells that have been induced to
differentiate to the required cell type (s). 2. On the surface of
the device as described above, a fibronectin or other extracellular
matrix or other adhesive layer may be deposited or printed in order
for the cells to attach to the surface and stay viable. 3. The
required number of cells (depending on the surface area and cell
type) may be plated on the substrate as described above, and
maintained in a healthy viable condition in the right culture
medium, to measure certain cell-specific variables, like ion
channel activity or electric action potential. 4. Sometimes an
additional or repeated action(s)/procedure(s) may be required to
further develop the disease model, sometimes this may entail either
one-time, continuous, or repeated stretching of the cells, to
100-200% or more of their original length, either in plane or in
such a way (usually) that they stretch out of plane, mimicking the
required disease situation. Sometimes there are shorter or longer
cycles of stretching followed by a defined period of relaxation.
During this process the action potential of the cells is measured,
and interpreted with respect to the activity of the one or more
membrane ion channels involved in generating ion fluxes that
generate electrical potential changes. 5. One or more
disease-specific variables are measured to prove that the disease
model resembles the actual disease to such extent that it can be
used for purposes of drug target discovery, drug development and
drug toxicity. 6. For purpose of drug target discovery, drug
development or drug toxicity the cells will be stretched in the
same ways as described under (4), in such a way that the stretch
occurs either in or (mostly) out of plane and the action potential
is continuously monitored and interpreted. During this procedure
chemical or biological compounds can be added that need to be
investigated. 7. After finishing the stretching part of the
experiment, several other measurements can be performed on the
cells, like DNA/RNA/protein/metabolyte analysis, to investigate the
action of the added compounds or other change in the cell
environment, like a pH or electrolyte, glucose, oxygen or
nutritional supplement or metabolite concentration change, on the
function/activity/viability of the cells.
[0101] Disease models that can in this way be created can be
divided in several categories, dependent on:
[0102] the percentage of stretch required, this is determined
either by the physiological stretching that occurs in vivo. For
example during heart beating when cardiomyocytes and endothelial
cells are stretched to a physiologically level, dependent on the
cardiac output/blood pressure with every beat; or
pathophysiological stretching like for example occurs in a tumor or
intracerebral bleeding or subdural hematome.
[0103] the level to which the stretch takes place out-of-plane. For
example for a model to simulate a beating heart the cardiomyocyte
cell layer must be pushed out-of-plane during the stretch, and
returned in plane during the relaxation part of the heart beat. If
the model for example mimics heart failure, the cardiomyocytes are
more extensively stretched out of plane to simulate increased
filling of the heart ventricle with blood, over the top in the
Frank-Starling curve. For stretch in a model for a growing tumor,
the stretch will be increasingly out of plane.
[0104] The manner in which the stretch is applied with time:
one-time short, continuous for longer period of time, or repeated.
In a cardiac or endothelial model the stretch is applied
repeatedly. For example, in a tumor model the stretch is continuous
and increasing. For example, in a model for intracerebral bleeding
the stretch is short and rapidly increasing to high/maximum
level.
[0105] the time period during which the stretch is applied: for
example intermittent (stretch-contraction cycles) with the
frequency of a heart beat under different conditions; or with the
stretch slowly increasing over hours in the case of a model for
subdural hematoma in the brain; rapid increase in stretch within
hours in the case of a model for intracerebral bleeding, very slow
in case of a model for a brain tumor)
[0106] the type of adhesion between the individual cells. The
extent to which stretching decreases the adhesion between cells
depends on the strength of the adhesive molecular bonds and the
cellular processes that regulate these bonds. For example in case
of a model for cancer, the level of stretch required to simulate
certain pathogenetic changes in the cancer cells will be tuned to
the strength of the intercellular adhesions, which is likely to be
specific for different tumors, such that the electrical resistance
over the cell layer is decreased.
[0107] The method for developing disease models as described above
is very suitable for those diseases that are caused by or modified
by stretching of cells, resulting in abnormal action potential or
functioning of ion channels. Examples are endothelial diseases like
atherosclerosis and hypertension and migraine; neurological
diseases like whiplash, concussion, brain tumors, intracerebral
bleeding and subdural hematoma; muscle diseases like Duchenne, and
gastrointestinal disease like irritable bowel syndrome.
[0108] It should be noted that the above-mentioned embodiments
illustrate rather than limit the invention, and that those skilled
in the art will be able to design many alternative embodiments
without departing from the scope of the appended claims. In the
claims, any reference signs placed between parentheses shall not be
construed as limiting the claim. The word "comprising" does not
exclude the presence of elements or steps other than those listed
in a claim. The word "a" or "an" preceding an element does not
exclude the presence of a plurality of such elements. The mere fact
that certain measures are recited in mutually different dependent
claims does not indicate that a combination of these measures
cannot be used to advantage.
* * * * *