Patent Description:
In the field of drug research, many drugs are tested on cardiotoxicity. Many (new) drugs affect the heart muscle contraction and profile, thus giving toxic effects. Drug-induced cardiotoxicity is a major adverse effect that has been encountered for some clinically important drugs. This toxicity has previously led to the post-marketing withdrawal of numerous pharmacologically active drugs and limits the efficacy of other clinically useful ones. Almost <NUM>% of drugs in the last four decades have been recalled from the clinical market worldwide due to cardiovascular safety concerns.

Drug-induced cardiotoxicity is an important cause of rejecting compounds in preclinical and clinical development. It represents one of the most serious side effects associated with novel drug development, and it is known to be one of the major toxic effects induced by several types of drugs. Assessing drug-induced cardiotoxicity risk including QT interval prolongation is considered nowadays an integral part of the standard preclinical evaluation of new chemical entities.

Cardiotoxicity may be tested in animal models, for example in rats. However, these models have drawbacks and do not always predict the effect in humans well. This may result in discarding potential beneficial medication, but also in accepting potentially toxic medication in a field trial on humans.

Another way to assess cardiotoxicity is to test the effect of the drug in vitro. The drug is added to an assembly of cardiomyocytes, which are grown in vitro. Afterwards, the effect to electrical stimuli is studied.

An example of such state of the art device is the HeartDyno (Trade Mark) device. This device consists of an oval shaped well, with two small pillars at the bottom. These devices are produced using thin film technologies. The mold is made via SU-<NUM> photolithography on a wafer, resulting in <NUM> deep features. PDMS is cast over these features and cured. After removal of the PDMS from the wafer, samples of <NUM> diameter are punched.

These samples are for example placed inside the wells of a <NUM>-well plate and glued to the bottom using silicone glue. A mixture of cardiomyocyte cells, cardio fibroblast cells, collagen, DMEM, NaOH and matrigel is added to the wells. The cardiomyocyte tissue is formed over several days. The tissue shows spontaneous contraction, but also contracts during electrical stimulation. During contraction, the two pillars deflect and the deflections are analyzed using video analysis algorithms.

<CIT> discloses a device related to generating 3D cardiac tissues. <CIT> discloses microfabricated arrays of micro-wells within a PDMS mould for culturing tissue.

Gluing the devices inside a well of a well plate is a cumbersome process. It is difficult to do this in a sterile way, and it requires extra manufacturing steps. Furthermore, the devices need to be aligned in such way that the pillars are oriented in the same way in each well, which requires extra care. A proposed solution is to 'clamp' the device inside the well of a well cell plate. This can be done by producing a device from an elastomeric material, with a slightly larger outer diameter than the well, so it will be kept in place by shear stresses. However, in this way, the negative space of the device will deform and this may render it unusable.

According to examples in accordance with an aspect of the invention, there is provided a device for testing in vitro muscle tissue, the device comprising:.

In vitro cardiac muscle tissue can be electrically stimulated to contract in order to simulate the behavior of in vivo cardiac tissue. The tissue can be grown between and around the two pillars such that, when the tissue contracts, the two pillars deflect. The displacement of the pillars caused by the deflection can be measured to determine the force from the contraction of the tissue.

Typically, the devices for testing in vitro muscle tissue are put into a well of a microwell plate. In order to keep the device in the well, the outer periphery of the test unit can be made slightly larger than the diameter of the well such that the shear stresses keep the device in place. However, this could cause the inner diameter of the wall section to deform.

In order to avoid or reduce the inner wall surface deformation, rib elements are added to the wall section to avoid or reduce the inner wall surface from deforming whilst keeping the shear stresses high enough to keep the device in place during use of the device without the use of glue. The outer diameter of the rib elements (at the outer periphery of the device) may be larger than the outer diameter of the base section in order to further reduce the deformation of the device.

The pillars may be taller than the wall section or the wall section may be taller than the pillars. Alternatively, the pillars and the wall section have the same height. Similarly, the wall section may have the same height as the rib elements, or the rib elements may have a different height from the wall section (e.g. taller or shorter). Each rib element may have the same height or a different height.

There may be two, three, four or more rib elements extending in at least two non-parallel directions within a virtual plane comprising the base section. However in one preferred example, there are four rib elements. A symmetrical design has the rib elements extending radially at <NUM> degrees apart from each other.

However, an asymmetrical design may be used with two pairs of diametrically opposite rib elements, but with the two pairs not orthogonal to each other (so making a non-perpendicular cross).

This forms a cross shape of the rib elements to provide a structure which would not deform under shear stress from any direction.

The inner wall surface may be elliptic or even rectangular, hence a shape with a non-unity aspect ratio.

This non-unity aspect ratio encourages the tissue to grow in a contraction direction in line with the separation gap between the pillars.

The contraction direction is the direction in which the tissue contracts. Thus, by having the contraction direction in line with the spacing between the pillars, the tissue will deflect the pillars towards the center point between the two pillars.

The height of the rib elements may be at most the same height as the height of the wall section. The height of the wall section may be at most <NUM>.

The height of the pillars may be at least <NUM> and at most <NUM>.

The device may be manufactured based on:.

The pillars are bulk manufactured using on of these methods in order to reduce the spread in pillar stiffness from the manufacturing method and increase the production rate, when compared to hand casting.

The device (or only the pillars, the base section and/or the rib elements) may be manufactured from a silicone rubber. The silicone rubber may comprise Elastosil <NUM>/<NUM>.

The pillars may be tapered towards the distal end, and the distal end of the pillars may be rounded or partly rounded.

Tapering the pillars allows the demolding step (of the manufacturing process) to be more reliable. Similarly, a rounded distal end of the pillars further increases reliability in the demolding step.

The Young's Modulus of the material used to manufacture the pillars may be at least <NUM> MPa and at most <NUM> MPa. For example, the Young's Modulus is between <NUM> MPa and <NUM> MPa. The stiffness of the pillars will depend on the Young's Modulus and the geometry of the pillars.

However, there are other material properties which are also relevant for the function of the pillars. The tensile strength (e.g. <NUM> N/mm<NUM>) and the max elongation (e.g. at break <NUM>%) are two of such relevant properties. The combination of the these properties makes it possible to demold the pillar structures with an aspect ratio near <NUM> (or even higher) and also for allows the demolding of mushroom geometries.

Thus, using injectable molding grades of silicone gives reliable end properties after manufacturing with easy scale up possibilities and is also suitable for geometries with an aspect ratio of <NUM> or higher for releasing from industrial molds. This is not possible with other casting solutions.

The invention also provides a set of devices for testing in vitro muscle tissue in a microwell plate wherein the set of devices is manufactured using multi cavity injection molding, wherein each cavity corresponds to one device.

Multi injection molding would increase the production rate of the devices, as well as ensuring an adequate mold flow and wall thickness ratio.

The invention also provides a system for testing in vitro muscle tissue, the system comprising:.

For example, the devices could be equally spaced on the array base in a rectangular array. This allows multiple devices to be manufactured without the need of a multi cavity injection molder.

The system may further comprise a microwell plate with a plurality of wells, wherein the plurality of devices on the array base are situated such that each device fits within a respective one of the wells.

For example, using an open bottom microwell plate, the system with the array of devices could be placed under the microwell plate such that the devices all fit within the wells of the microwell plate. The system would be manufactured to fit within the microwell plate (i.e. the outer diameter of the devices would fit within the wells and the spacing of the devices would be the same as the spacing between the wells).

The muscle tissue can then be grown in the well of the microwell plate around the pillars of the device and within the wall section.

The devices may be manufactured of elastic material and the outer periphery of each device may be larger than the inner periphery of the wells of the microwell plate by, at most, <NUM>%.

If the devices have a slightly larger size than the wells and are manufactured from a flexible material, the devices will be able to fit within the well, but will provide a shear stress to the device due to the difference in diameter. The difference in diameter is dependent on the material used to manufacture the devices, the shear stress required to keep the device in the well during muscle contractions etc..

The system may further comprise a pair of electrodes for stimulating muscle tissue.

The invention also provides a method of manufacturing a device for testing in vitro muscle tissue, the method comprising:
injection molding the device using a mold which defines:.

The invention provides a device for testing in vitro muscle tissue, for example in a microwell plate, the device comprising a base section and two pillars, each protruding from the base section. The device also comprises a wall section with an inner wall surface surrounding the pillars and an outer wall surface, wherein the wall section comprises at least two rib elements extending outwardly from the outer wall surface to an outer periphery of the device.

<FIG> shows a representation of a device <NUM> with two tapered pillars <NUM>. The pillars <NUM> are made of flexible material, the shape of the pillar <NUM> is designed for mass production and the shape and height of the pillars <NUM> are adjusted to have the required material properties for testing samples of tissue <NUM>. The design of the pillars <NUM> is adapted to ensure demolding. The pillars <NUM> are tapered, with a wider base and smaller top.

The pillars <NUM> extend from a base section <NUM> and are enclosed within the inner surface of a wall section <NUM>. The pillars <NUM> are designed such that cardiac tissue <NUM> can grow between and around the pillars <NUM>. The cardiac tissue <NUM> can then be contracted with electrodes and the contractions of the cardiac tissue <NUM> from the electrical signal can be measured by the displacement of the pillars <NUM>.

<FIG> shows a cross section of the device <NUM> with cardiac tissue <NUM> between the pillars <NUM> with no contraction. <FIG> shows the same device <NUM> but with the cardiac tissue <NUM> contracting. The pillars <NUM> are displaced due to the contractions of the cardiac tissue <NUM>.

<FIG> shows the same device <NUM> from a top down view when the cardiac tissue <NUM> has not contracted. In this example, the inner wall surface is elliptical. By growing cardiac cells in the ellipsoid cavity the tissue <NUM> forms a direction for contraction in line with the spacing between the pillars <NUM>.

The device <NUM> could be manufactured by injection molding, 3D printing, urethane casting or press casting. The tapered pillars <NUM> allow a more robust demolding method for injection molding, urethane casting and press casting and also allow a more stable structure to be printed from 3D printing methods. These methods allow mass production of the devices <NUM>. The following examples will be described in connection with injection molding. However, any feature or advantage disclosed may also apply to other manufacturing methods.

In this example the base section is shown at the bottom. However, this is not always the case and the base section may have any orientation.

The pillars <NUM> may have a cuboid shape and be tapered on one, two, three or four of their sides. For example, two sides of the pillars <NUM> (e.g. facing inwardly towards the tissue and facing outwardly away from the tissue) may be tapered and the other two sides may be perpendicular to the base section <NUM>. The exact taper for a pillar <NUM> will depend on the dimensions of the pillar <NUM> itself but may range from a <NUM> degree taper to a <NUM> degree taper relative a perpendicular line from the base section <NUM>.

The pillars <NUM> may instead be cylindrical and as such the one side would be tapered. Similarly, the exact taper for a pillar <NUM> will depend on the dimensions of the pillar <NUM> itself but may range from a <NUM> degree taper to a <NUM> degree taper relative to a perpendicular line from the base section <NUM>. An example taper would be between <NUM> degrees and <NUM> degrees.

Additionally, the distal end of the tapered pillars <NUM> from the base section (i.e. the tip of the tapered pillars) may also have a head section. The pillar head would have a larger diameter (in one or two dimensions) than the rest of the pillar <NUM> and would stop tissue from sliding towards the distal end of the pillars and disconnecting itself from the pillars.

For a device <NUM> with a head section which is injection molded, the pillars <NUM> (beneath the head) are again preferably tapered. The tapering of the pillars reduces the friction between the pillars <NUM> and the mold when demolding the devices <NUM>. The tensile strength (e.g. <NUM> N/mm<NUM> to 11N/mm<NUM>, preferably <NUM> N/mm<NUM>) and the maximum elongation (e.g. at break <NUM>% to <NUM>%, preferably <NUM>%) of the material (i.e. silicone rubber) used to manufacture the pillars make it possible to demold the pillar structures with a head section.

Due to the reduced friction between the tapered pillars <NUM> and the mold, the only significant force required to remove tapered pillars <NUM> with head sections is the force required to elastically deform the pillar head to fit through the smallest section of the mold. The properties (e.g. tensile strength and maximum elongation) of the silicone rubber allow it to deform elastically in such a manner and thus retain the original shape once it has been removed from the mold. Other materials with similar properties could also be used.

The shape of the head section can also be designed to reduce the force required to pull it out of the mold. The shape of the head section will also depend on the properties of the material, the shape of the pillars <NUM> and the taper used on the pillar. For example, the head section could be a rounded shape, for example even spherical with a diameter larger than (or equal to) the width of the largest side of the pillars <NUM>. The pillars with a head section could also have a mushroom-like geometry.

Thus, using injectable molding grades of silicone gives reliable end properties after manufacturing with easy scale up possibilities and is also suitable for tapered pillars <NUM> with a head section. This is not possible with other casting solutions.

<FIG> shows model pillars <NUM> for calculating the displacement of the tapered pillars <NUM> due to forces applied. The tapering of the pillars <NUM> influences the stiffness and elasticity of the pillar <NUM> and this should be compensated. In translating the design into an injection molding design, changes in material and geometry should be compensated to keep correct stiffness and elasticity of the pillars <NUM>. The influence of changing the design of the pillars <NUM> design can be calculated with simulation techniques.

A common soft material for injection molding is Elastosil <NUM>/<NUM>, with shore hardness 40A. Producing the device <NUM> with this material influences the elasticity characteristics of the pillar <NUM>. Simulations have been performed to estimate these effects, and to propose an optimized design.

Using injection molding increases the reproducibility of the stiffness, and the hardness is much better. The silicone from Sylgard <NUM> is not so reliable due to temperature variations of the curing, in combination with poor accuracy of the mixing (and hence weight differences) of the two components in a desired, e.g. <NUM>:<NUM>, ratio for the silicone A and the cross linker B. For injection molding, components A and B are mixed in a <NUM>:<NUM> ratio in much larger quantities and the stoichiometric mismatch is minimal. It can also be scaled up easily.

After injection molding, the reaction of the Elastosil <NUM>/<NUM> will initiate after heating up in the mold to 150C or higher. A problem with the Sylgard <NUM> is that it starts to cure at room temperature. The idle time is also low for Sylgard <NUM> so that it becomes stiff sooner, such that casting it in small cavities may not be possible. With injection molding, the mixture heats up in the heated mold and has a low viscosity at high pressure (i.e. 30MPa to <NUM> MPa), such that all the cavities for the pillars <NUM> will be filled.

<FIG> shows a simulation of tapered pillars <NUM> for measuring the strain on the pillars <NUM> when a force is applied. The typical elastic modulus of Sylgard <NUM> is <NUM> MPa and for Elastosil is <NUM> MPa. Bulk modulus of <NUM> GPa is used for both materials. A Neo-Hookian approximation is employed in the calculations.

<FIG> shows a simplified representation of the displacement measurement <NUM>. The measurement point <NUM> on the pillars <NUM> is at ¾ height. Devices using Slygard <NUM> have a stiffness of <NUM>/N at <NUM> displacement, so the stiffness of the Elastosil pillars <NUM> with respect to the Sylgard <NUM> pillars is estimated at <NUM> displacement.

In the simulation of <FIG>, a distributed force is applied at the defined area of a pillar <NUM> and an area between ½ and ¾ of the pillar <NUM> height is subjected to this force. The force is increased until the horizontal displacement of the monitoring point (at ¾ height of the pillar <NUM>) reaches <NUM>. The pillar <NUM> heights are <NUM>.

The stiffness of the Sylgard <NUM> pillars is derived at <NUM> displacement, and compared to the stiffness of the Elastosil <NUM>/<NUM> pillars <NUM>. The stiffness of the tapered pillar design is about <NUM> times higher than the Slygard <NUM> pillars. This can be addressed with a new pillar shape (i.e. higher pillar <NUM>, different tapering angle, etc.). The choice of stiffness will depend on the needs of any particular user.

For example, higher pillars <NUM> can be used to compensate for the changed stiffness so that the pillars <NUM> can be produced with injection molding techniques, making the design suitable for mass production.

<FIG> shows simulated pillars <NUM> with tapered and rounded tops. <FIG> shows a pair of pillars <NUM> with a height of <NUM>. <FIG> shows a pillar <NUM> with a height of <NUM>.

The rounded top design is <NUM>% stiffer than the reference configuration shown in <FIG>. However, because this can be mass manufactured, the spread in stiffness will be much lower. To compensate completely for the stiffness change, pillars <NUM> of <NUM>-<NUM> height have been found to be suitable. The rounded top pillars <NUM> are better able to distribute local stresses. The rectangular pillars <NUM> have a higher stress concentration which gives higher variations.

A mold for a device <NUM> with tapered and rounded tops has been designed and made, and devices <NUM> have been made from this mold. Demolding of <NUM> pillars <NUM> in Elastosil <NUM>/<NUM> material was found to be successful.

The mold has a base portion for defining an outer wall (the underside) of the base section <NUM> and an outer wall (the radially outward facing outer wall) of the wall section <NUM>. A top portion is for defining an inner wall (the top side) of the base section <NUM>, the inner wall (the radially inwardly facing wall) of the wall section <NUM> and the pillars <NUM>, wherein the top portion comprises two cavities for forming the two pillars <NUM>.

The cavities were fitted with an insert to define the pillars, so the shape can easily be changed to answer varying product requirements. Furthermore, this way of production ensures a well defined top shape of the pillars <NUM>. Different inserts could be used in the cavities to produce different pillar shapes (and thus different stiffness values).

By growing cells in the ellipsoid cavity, the muscle tissue <NUM> forms a direction for contraction in line with the spacing between the pillars <NUM> (i.e. parallel to a line joining the centers of the two pillars), but initially the cells may not attach to the pillars <NUM>. Uniform cell proliferation on the extracellular matrix in this device <NUM> into a confluent layer of cells to form muscle tissue <NUM> is a critical step.

To solve this, silicone pillars <NUM> can be modified with carboxylic groups so that direct cell attachments to the pillars <NUM> is possible. By bulk modification of silicone with linoleic acid, carboxylic groups are introduced and active on the surface so that cell can adhere onto the pillars <NUM>. In this way cardiomyocytes can better grow into muscle tissue <NUM> between the two pillars <NUM>.

<FIG> shows a 3D representation of the device <NUM> with pillars <NUM>. In this example, the inner surface of the wall section <NUM> has an elliptical shape. The device <NUM> can be described as having a cavity with two pillars <NUM> within the cavity (i.e. the cavity is defined by the wall section <NUM>).

Additionally, a pillar head could be added to the distal (top) end of the pillars <NUM> to avoid the muscle tissue <NUM> from disconnecting from the pillars <NUM> due to the movement of the muscle tissue <NUM> caused by the contractions. The elasticity of the material will allow the pillars <NUM> to be demolding despite the pillar head if suitable dimensions are chosen.

<FIG> shows a top down view of a device <NUM> manufactured with injection molding. Marking from the injection molding process can be seen near and around the pillars <NUM>. Similarly, in this example the inner surface of the wall surface <NUM> is elliptical.

<FIG> shows a device <NUM> with cardiac muscle tissue <NUM>. The cavity was filled with gel (Extra Cellular Matrix) and the cardiomyocytes grew on top of the gel into muscle tissue <NUM>. When the tissue <NUM> contracts, the pillars <NUM> will move and the muscle function can be monitored when the tops of the pillars <NUM> are provided with suitable indicators (e.g. colors applied to the top of the pillars to enable more precise focusing of a microscope).

<FIG> shows the results for a first experiment. The graph shows a plot of beating frequency (y-axis) versus pacing (stimulation) frequency. The pacing frequency is the frequency of the electrical signal used to stimulate the muscle tissue <NUM> and the beating frequency is the frequency at which the muscle tissue <NUM> is observed to contract.

The experiment involved stimulating cardiac muscle tissue <NUM> in the device <NUM> and comparing the result to other devices. The formed tissue <NUM> was electrically stimulated with 35V, a pulse duration of <NUM> and a range of pacing frequencies with increments of <NUM>. The muscle contraction force was calculated using the following equation: <MAT>.

Equation <NUM>: where: F = Force, E = Youngs' Modulus, b = pillar length, h = pillar width, x = position of tissue on pillar in z-direction, L = height of the pillar, δ = displacement.

The results obtained are shown in <FIG> shows the beating frequency (y axis) as function of the pacing frequency (x axis) for five tapered pillar devices <NUM> manufactured with injection molding. <FIG> shows the beating frequency (y axis) as function of the pacing frequency (x axis) on five HeartDyno devices.

It is observed that the tapered devices are on a par with the HeartDyno devices or perform even better due to the cardiomyocyte tissue <NUM> responding more consistently to pacing at a higher frequency. The tapered devices used for the results in <FIG> were also rounded.

<FIG> shows the results of a second experiment. The experiment again involved stimulating cardiac muscle tissue <NUM> in the device <NUM>. <FIG> shows the beating frequency (y axis) as function of the pacing frequency (x axis) of seven tapered pillar devices <NUM>. <FIG> shows the normalized beating frequency (y axis) against the pacing frequency (x axis) for the seven tapered pillar devices <NUM>. It can be seen that the contraction amplitude goes up, which indicates a positive force-frequency relationship. This is a hallmark of mature cardiomyocytes.

These results show that the tapered pillar devices <NUM> perform on a par or slightly better than the HeartDyno devices. Furthermore they have the advantage that the stiffness spread is lower due to the use of mass production techniques. Also the cost can be lower due to the use of mass production techniques.

<FIG> shows a top view of a device <NUM> (pillars <NUM> not shown). <FIG> shows a device <NUM> inserted in the well of a microwell plate (i.e. well cell plate) adequately. <FIG> shows a device <NUM> when it is deformed due to being inserted in the well of a well cell plate inadequately. This deformation can occur if the device is made slightly larger than a well in a well cell plate (to avoid using glue).

The deformation is exaggerated in <FIG> and may be caused after the device <NUM> is positioned in a non-ideal well shape causing unpredictable deformation of the inner surface and thus influencing part to part differences in cell growth performances and reproducibility.

<FIG> shows a device <NUM> with base section <NUM>, wall section <NUM> and rib elements <NUM> around the outer perimeter of the device <NUM> (pillars <NUM> not shown). This makes it possible to use a slightly larger outer diameter of the device <NUM>, compared to the inner surface of the well of a well cell plate, while not deforming the inner surface of the device <NUM>. In this way, the shear stress of the device <NUM> against a well cell plate can be increased to such a point that gluing the device <NUM> in the well cell plate is no longer necessary. Thus, the rib elements <NUM> (in this case, cross ribs) can increase the shear stress and reduce deformation of the device <NUM>, in particular, deformation of the inner surface.

The device <NUM> is made of an elastomeric material, which can be deformed upon application of an external force. Designing cross ribs <NUM> around this wall section <NUM> stops the inner wall surface from deforming when inserted in a well, while keeping the shear forces high enough to avoid any form of adhesive needed between the device <NUM> and the well.

Additional benefits besides eliminating the necessity to use glue include that the flexible material of device <NUM> and integrated cross ribs <NUM> will overcome tolerance issues of the shape of the device <NUM> or tolerances of an array (part to part variations are overcome). In some manufacturing methods, the demolding direction of either the device <NUM> and/or the well cell plate can help to increase shear forces or provide a complete mechanical lock.

In this example, devices <NUM> with four cross ribs <NUM> are shown. However, the devices <NUM> could also have two cross ribs <NUM>, three cross ribs <NUM> or more than four cross ribs <NUM>.

<FIG> shows a device <NUM> with rib elements <NUM> manufactured with injection molding. The device <NUM> is made via injection molding of Wacker Elastosil LR <NUM>/<NUM> (shore hardness A <NUM>). The device <NUM> is punched with a <NUM>-mm-punch and can be placed in a well of a well cell plate (e.g. with <NUM> wells), with an inner diameter of <NUM>.

It has been found that the shear stress of the devices <NUM> inside the well of the well cell plate is high enough to keep the devices <NUM> in place, during a complete protocol of growing and testing cardiac tissue with a duration up to <NUM> days.

<FIG> shows an array <NUM> of devices <NUM> for integration in a well cell plate. Besides single devices <NUM>, the devices <NUM> can also be produced as an array <NUM> integrated on a slab of material. For this purpose, a slab of material is produced, with an array of devices <NUM> with cross ribs <NUM>. This slab can be placed under a well cell plate with an open bottom. In this way, the array <NUM> closes the well cell plate and provides the devices <NUM> for cell culture. The array <NUM> can be circular, rectangular or any other ratio in between depending on the requirements.

<FIG> shows a <NUM> well cell plate with an open bottom. This well cell plate can be used for the array <NUM> of devices shown in <FIG>. Alternatively, devices <NUM> in an array <NUM> integrated onto a material slab can also be directly used without the need of a well cell plate. In this case, the outer walls define the devices <NUM> of the array <NUM> directly over a shared base, and this structure can be produced in one piece. The outer walls will act as an alternative to the wells in the well cell plate.

<FIG> shows a plurality of devices <NUM> manufactured with multi cavity injection molding.

Producing one slab material (devices in an array <NUM>) requires a big injection molding machine to fill the large volume and the small details of the pillars <NUM>. Injection molding works best with even wall thicknesses (e.g. thickness of the wall and of the pillars <NUM>). If they differ by a large ratio, small details will freeze more rapidly even when the mold has not been filled completely and large volumes will freeze by polymerization the slowest. An unwanted situation may occur if the smallest details are frozen already while the bigger volumes still need to be filled. This will cause defects and, in this case, the risk that the mold part for the pillars <NUM> is not filled completely.

This issue makes it difficult to produce the small details (i.e. the pillars <NUM>) in the large slab. This can be solved by balancing the amount of material and wall thickness. <FIG> shows several separate devices <NUM> each with an optimized wall thickness and flow path. Less volume to freeze also improves production cycle times.

By manufacturing separate devices <NUM>, tolerances differences within well cell plates can be overcome compared to an array <NUM> of devices <NUM> with pillar details. Producing an array <NUM> may lead to leakage in the well plate due to tolerance and shrinkage differences whereas producing separate devices <NUM> can overcome this. The devices may be manually or robotically positioned into e.g. a <NUM>-well cell plate or any other target.

One problem that may be encountered with these devices <NUM> is knowing the exact height of the muscle tissue <NUM> on the pillars <NUM>. As shown before (in equation <NUM>), the displacement of a pillar <NUM> is dependent on the height of the muscle tissue <NUM> on the pillar <NUM>. Also, this position can change during experiments. The muscle tissue <NUM> is known to creep up the pillars <NUM> due to mechanical forces. Furthermore, during pillar deflection, the muscle tissue <NUM> can come off of the pillars <NUM>, rendering the experiment unsuccessful.

In order to avoid this problem, the device <NUM> may be "inverted", such that the pillars <NUM> protrude downwards (relative to a well cell plate).

<FIG> shows an inverted pillar device. The inverted pillar device has two pillars <NUM> protruding from the base section <NUM>. The inverter pillar device is fitted on a so-called ultra-low attachment well in a well cell plate. The pillars 'hang' in the well and muscle tissue <NUM> can be grown around them. Pillar <NUM> movement analysis can take place from below the well (if the well call plate is made of a transparent material).

A cell/collagen mixture is deposited on the bottom of the ultra-low attachment well. The base section <NUM> is placed on top of the well and acts as a lid for the well while the pillars <NUM> project into the cell/collagen mixture. When the cardiomyocyte tissue <NUM> forms, it grows around the pillars <NUM>. It will not adhere to the well, because an ultra-low attachment well is used. When the cells start to contact each other, the pillars <NUM> will begin to move. This motion is captured with a microscope placed below the well.

In the inverted pillar device, the cell culture is always at the end of the pillars <NUM>, thus the height position of the muscle tissue <NUM> is well defined. This makes the forces required for deflecting the pillars <NUM> more reproducible. Also, upon movement of the pillars <NUM>, the muscle tissue cannot detach itself from the pillars <NUM> due to gravitational forces. Furthermore, the relative position of the two pillars <NUM> is defined and will not add additional spread.

An additional advantage of this example is that the inverted pillar device can again be mass produced via injection molding. Also, the range of possible material stiffness values is larger, because the pillars <NUM> hang from the base section <NUM>. In an upright device <NUM> design, the pillars <NUM> need a certain stiffness to keep standing straight.

The measurement system may comprise a microscope underneath the well cell plate looking up. This is comparable to current systems for measuring pillar displacement. In either system, putting something on the bottom of the well will interfere with the measurements.

In this inverted design, the base section can functions as a plug for the well, avoiding spill or sealing the well from contaminants (e.g. air).

Cardiomyocyte muscle tissue <NUM> can be paced as mentioned above. This is implemented using a voltage of, for example, <NUM> V at a certain frequency (e.g. simulating heart beats). The cardiomyocytes will contract upon stimulation. This contraction can be monitored, while adapting the pace frequency. In order to pace the cells, two electrodes <NUM> are placed in the well.

These electrodes can be integrated in the inverted device as shown in <FIG>. In this way, positioning the electrodes does not hamper the field of view for cell visualization from the bottom of the well. Furthermore, the electrodes <NUM> are always positioned adequately as they can be held by the base section <NUM> and the well does not have to be opened (i.e. the base section <NUM> does not have to be removed) in order to stimulate the muscle tissue <NUM>. This prevents contamination of the cell culture.

For the conventional upwardly projecting pillars, it is of interest to be able to determine the height of the muscle tissue <NUM> up the pillars.

<FIG> shows a device <NUM> with a staircase like structure <NUM> located outside the cavity area. The staircase structure <NUM> is used to determine the height positioning of the muscle tissue <NUM> on the pillars <NUM>. The staircase in one example has steps of <NUM> each, but this dimension can of course be changed depending on the size of the device <NUM>, the pillars <NUM> and/or the cell well plate. For example, if the device <NUM> with a structure <NUM> is manufactured using injection molding, the step size can be changed by using a different injection molding insert.

When using a microscope to view the muscle tissue <NUM>, the microscope must be focused on the muscle tissue <NUM> itself. When focusing on the muscle tissue <NUM> on the pillars <NUM>, the z-position (i.e. height) can be determined by looking at the corresponding steps of the staircase structure <NUM> which are also in focus. Each step of the staircase structure <NUM> can be marked (e.g. with numbers or letters or other characteristic feature) to easily read out the corresponding z-position. The height of the muscle tissue <NUM> can then be determined and thus the forces on the pillars <NUM> can be normalized through calculation (i.e. using equation <NUM>).

In this example, the wall section <NUM> has a separate outer surface for the structure <NUM> outside the outer wall which defines the cavity. The structure <NUM> can instead be placed within the inner surface of the outer wall <NUM>. The height determination structure may then be on the inside of the wall <NUM> close to the pillars so easily visible.

<FIG> shows a plan view of the device of <FIG>.

<FIG> shows a device <NUM> with a conical structure <NUM> for the height determination. In this example, the diameter of the cone changes with the height of the structure <NUM>. Thus, the diameter of the cross section of this cone which is in focus when using a microscope corresponds to the z position of the muscle tissue <NUM>. In this example there are no discrete steps in measurements of height (as experienced with the staircase structure) and as such the height can be measured at any position.

The structure <NUM> could be any other shape in which the cross section of the structure <NUM>, when viewed from a top down view, changes relative to the height of the structure <NUM> from the base section <NUM> (e.g. tapered 3D shapes, step based shapes etc.).

The structure <NUM> may be taller than the pillars <NUM> and/or the wall section <NUM> or it may be the same height as either of them. In the case that the structure <NUM> is taller than the pillars <NUM>, the structure <NUM> could have a height mark on it which would indicate the height of the pillars <NUM> (or a height slightly shorter than the height of the pillars) such that it can act as a warning mark for the user of a microscope to be aware that, if the muscle tissue <NUM> and the height mark are both in focus, the muscle tissue <NUM> may be in danger of disconnecting from the pillars <NUM>.

Alternatively, the structure <NUM> may be shorter than the pillars <NUM>. In this case, once the user of a microscope can no longer focus on the structure <NUM>, this may act as a warning sign that the muscle tissue <NUM> may be in danger of disconnecting from the pillars <NUM>. There may also be a plurality of height marks on the structure <NUM> at different height locations to make the height measurements easier.

The previous examples have been described using a microscope to image the pillars <NUM> and thus detect and measure the displacement of the pillars <NUM>. However, any other imaging system capable of measuring the pillars <NUM> may also be used (e.g. cameras etc.).

Claim 1:
A device (<NUM>) made of elastomeric material for culturing in vitro muscle tissue (<NUM>), the device (<NUM>) comprising:
a base section (<NUM>);
two pillars (<NUM>) each protruding from the base section (<NUM>); and
a wall section (<NUM>) with an inner wall surface surrounding the pillars (<NUM>), and an outer wall surface, characterised in that:
the wall section (<NUM>) comprises at least two rib elements (<NUM>) extending outwardly from the outer wall surface to an outer periphery of the device (<NUM>).