Optical reading device with a cell culture unit capable of culturing contractile tissue

Abstract

According to one aspect of the present invention, an optical reading device is provided, the optical reading device comprising a cell culture unit capable of culturing contractile tissue, the cell culture unit being provided with an elastic tissue support structure, the elastic tissue support structure extending into the cell culture unit such that the contractile tissue exerts a contractile force on the elastic support structure to generate a deformation of the elastic support structure, the reading device further comprising one or more light generators and light sensors formed as or including optical elements for deflecting light from the light generator by the elastic support structure onto the one or more light sensors such that deformation of the elastic support structure causes the light to move above the light sensors, and a controller for aligning the movement of the light above the light sensors to measure the contractile force of the contractile tissue.

Description

[Technical Field]

[0001] The present invention relates to the technical field of optical reading devices comprising a cell culture unit capable of culturing contractile tissue. [Background technology]

[0002] In particular, the present invention relates to an optical reading device comprising a housing for receiving a test plate provided with cell culture units on which a contractile tissue can be formed according to a fixed pattern, the contractile tissue being able to float inside the culture unit. In the prior art, the cell culture units are provided with elastic support structures extending within the cell culture unit such that the contractile tissue exerts a contractile force on the elastic support structure to generate a deformation of the elastic support structure.

[0003] One challenge with this system is the difficulty of generating large-scale test environments because the system requires detailed optical study of the mechanical interactions between the tissue and supporting structures, for example, by using imaging, particularly video confocal microscopy. This is because, in typical situations, shrinkage can be a few micrometers or less, making it difficult to study. Therefore, the structures must be imaged and the images studied. This type of imaging is cost-intensive and labor-intensive. Therefore, current solutions have low throughput, provide only snapshots, and / or may have a negative impact on drug testing.

[0004] It is an object of the present invention to provide an optical reading device that is easy to manufacture on an industrial scale at competitive costs. Summary of the Invention [Means for solving the problem]

[0005] This aim and these objects, as well as other objects that will become apparent from the following description and the accompanying drawings, are achieved according to the invention by a reflecting structure as claimed in claim 1 proposed below.

[0006] The optical reading device includes a cell culture unit capable of culturing contractile tissue. The cell culture unit is provided with an elastic tissue support structure extending into the cell culture unit such that the contractile tissue exerts a contractile force on the elastic support structure to generate a deformation of the elastic support structure. The reading device further includes one or more light generators and light sensors. The elastic support structure is formed as or includes an optical element to deflect light from the light generator by the elastic support structure onto one or more light sensors. In this manner, deformation of the elastic support structure causes the light to move above the light sensors. A controller aligns the movement of the light above the light sensors to measure the contractile force of the contractile tissue.

[0007] By virtue of the present invention, it is possible to provide an optical reading device which allows testing without the testing being disturbed by measurements. It should be noted that a resilient support structure of the type described above is known per se from WO2021206551. However, this is a system which is not suitable for imaging large areas.

[0008] Such an imaging system provides an optical reading device that can efficiently and easily monitor mechanical movements on a large scale.

[0009] Further features and advantages of the elastic support structure according to the invention will become more apparent with reference to the description given below and the accompanying drawings, which are provided purely for illustrative and non-limiting purposes. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows an exploded view of one embodiment of an optical reading device along with a schematic cell culture unit array. [Figure 2] 10A-10C show alternative embodiments of resilient support structures formed from optical fibers. [Figure 3]1 illustrates an exemplary manufacturing method for creating a support structure that is combined with an array of cell culture units. [Figure 4] 1 shows a support structure with a focusing lens. [Figure 5] 1 shows an exemplary deflection curve of a resilient pillar. [Figure 6] 10 illustrates an exemplary spot displacement of an elastic pillar. [Figure 7] 1 shows several support structures with optical elements. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description of the Drawings Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0012] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs, as read in the context of the description and drawings. It will be further understood that terms as defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and not in an idealized or overly formal sense, unless explicitly so defined herein. In some instances, detailed descriptions of well-known devices and methods may be omitted so as not to obscure the description of the present systems and methods. The terminology used to describe particular embodiments is not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural unless the context clearly dictates otherwise. The term "and / or" includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms "comprises" and / or "comprising" specify the presence of the stated features, but do not exclude the presence or addition of one or more other features. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0013] The optical readout device can be provided as a large-area optical imager, either as an integrated or freestanding device, to measure microscopic (biological) movements and derive mechanical parameters such as flow and / or contractile force (e.g., quantitative measurements during drug screening). In one embodiment, mechanically flexible micropillars act as optical fibers to guide light onto a pixel (or array of pixels), and deflection of such optical fibers upon external stimuli is translated into light intensity fluctuations or spot displacements. Note that high positional accuracy (better than the pixel pitch) can be obtained by interpolation using both intensity and spatial information.

[0014] While exemplary embodiments of the system and method are shown, alternative ways may be envisioned by those skilled in the art, having the benefit of this disclosure, to achieve similar functions and results. For example, some components may be combined or divided into one or more alternative components. Finally, these embodiments are intended to be merely exemplary of the system and should not be construed as limiting the scope of the appended claims to any particular embodiment or group of embodiments. Thus, while the system has been described in particular detail with reference to certain exemplary embodiments thereof, it should also be understood that numerous modifications and alternative embodiments can be devised by those skilled in the art without departing from the scope of the system and method as set forth in the following claims. Accordingly, the specification and drawings are to be considered illustrative, and not limiting, of the scope of the appended claims.

[0015] Preferably, the optical reading device has a size slightly larger than a standard culture plate, so that the reading device can be contained and read inside the incubator. As a result, real-time measurements can be performed without changing the conditions under which the cell culture is performed. Such a configuration allows for a compact design of the reader, which can be coupled in a planar manner, so that it can be installed entirely inside the incubator.

[0016] While different light guide structures can be contemplated, a convenient form of combining a resilient structure and a light guide is provided by an optical element designed as an optical fiber. The optical element may be a refractive or reflective element, so that light is refracted or reflected to form a light beam that is directed onto a light sensor. This somewhat magnifies the physical deflection of the structure, making it easier to measure. The resilient support structure may be provided on a support plate that is separate from the test plate and stacked on the test plate so that the resilient support structure protrudes into the cell culture units of the test plate. Having a separate support plate is a convenient way to provide an optically optimized structure that is suitable for growing contractile tissue thereon and to position the optical structure inside the culture units. For example, the support plate may further include through-holes for receiving fluid from a well plate to provide culture fluid to the cell culture units. A light generator may also be provided on the support plate. The support plate may further include optical fibers provided in a pattern corresponding to the cell culture units. Alternatively, the support plate may include a light-blocking layer on the support plate surface to allow light only to pass through the optical fibers. For example, the optical element may be a light-blocking element provided within the resilient support structure, whereby light is at least partially blocked by the light-blocking element, resulting in sensed alignment of the moving light. In a further embodiment, the housing includes a base plate within which the optical sensor is provided, the base plate having a shape such that it can be coupled to a test plate to provide optical contact. In a further example, the optical sensor is pixelated. In some embodiments, the optical element forms a sub-pixel sized light beam such that a moving spot modulates a single pixel signal. This provides very fast readout of small deviations, is made possible without much processing load, and is therefore suitable for further efficient scaling. In one embodiment, the optical sensor is formed by a pixelated organic photodetector. The resilient support structure may further include electrical conductors to provide electrodes for stimulating the contractile tissue.

[0017] In one embodiment, the resilient support structures may be spaced apart by a distance of 0.1 to 10 mm, preferably 0.1 to 5 mm, preferably 0.1 to 3 mm, preferably 0.1 to 2 mm, preferably 0.1 to 1 mm, for example about 0.6 mm or about 0.9 mm.

[0018] 1, an embodiment of the optical reading device 1 is shown in an exploded view, together with a schematic cell culture unit array 6. The optical reader 1 comprises a housing 2 with a flat test plate 3 provided with cell culture units 6. The cell culture units 6 of the test plate 3 form a fixed pattern, in the example of the drawing, in the form of a matrix of several wells, which is typical in practice, for example a 96-well plate. For clarity, only a limited number of wells are shown in the drawing. In a separate sensor part 7, the optical conversion element 4 is, for example, a photodiode or a different type of semiconductor sensitive in the relevant optical range.

[0019] The transducer elements 4 are provided in the sensor portion 7 in a pattern of the same type as the wells 6 of the test plate 3. The device according to the invention has a size that substantially corresponds to the size of a standard test plate, so that the reading device can be included in an incubator (not shown) for culturing biological material and can be read in situ without mechanical disturbances and in particular without the need to remove the plate from the incubator. To that end, the invention comprises a method for testing contractile tissue using an optical reader, comprising the steps of providing the contractile tissue in a test plate 3 comprising cell culture units 6, coupling the reader to the test plate, and inserting the reader into the incubator, wherein measurement signals from the reader are stored in a memory of the reader and / or output to a central processing unit.

[0020] On the test plate 3, coupling elements 8 can be present in the form of grooves, into which the test plate can slide and be fixed to form a fixed connection with the optical reader 1. Also, elastic support structures 15 are provided on the support plate 5, which is separated from the test plate 3 containing the cell culture units 6 and stacked thereon. In this way, the elastic support structures 15 protrude into the cell culture units 6 of the test plate 3. The elastic support structures 15 are suitable for supporting contractile tissue, so that the tissue can exert a contractile force on the support structure, causing it to deflect, as will be explained below. For this purpose, the support structures 15 include optical elements, which can have different embodiments, as shown in FIG. 5 . In the illustrated embodiment, the optical elements are designed as optical fibers, suitable for providing support for the tissue, for example by having a biocompatible cladding. Accordingly, the support plate 5 comprises optical fibers 15 arranged in a pattern corresponding to the cell culture units 6.

[0021] By means of connections 9, the optical transducers (for example formed by pixelated organic photodetectors) 4 are connected to an electronic control unit 10 for controlling the respective transducers 4, in particular for controlling the timing of reading the signals from the transducers. By means of external coupling, the control unit 10 can be connected to a computer as a peripheral device.

[0022] The reader 1 is suitable for analyzing light coming from wells 6 of a test plate 3. To that end, in the optical reader 1, a light generator 13 is provided on a support plate 5, but can also be coupled via other light coupling structures. In the illustrated embodiment, the light source portion support plate 5 comprises one or more light generators and light sensors in addition to an elastic support structure 15 formed as an optical element, for deflecting one or more light beams generated from the light generator 13 onto the light sensor of the sensor portion 7 by means of the elastic support structure. The reader can further comprise additional structures, for example a well plate 11. In the illustrated structure, the support plate 5 further comprises through-holes for receiving fluid from a well plate in order to provide culture fluid to the cell culture unit.

[0023] FIG. 2 shows an alternative embodiment of a resilient support structure formed as an optical fiber. In variant A, fiber 15 is part of a support plate 5 or other support structure. Tissue attached to fiber 15 exerts a contractile force F on it, for example, by having attachment to a portion of a well or other support element (not shown). Light I is injected into the fiber, resulting in a moving light beam O that can be detected as it moves over a light sensor that can detect lateral movement of the light beam O. In variant B, the fiber may be part of the cell culture unit 6, for example, by protruding through the bottom wall. The contractile force F results in a moving fiber into which light is injected at I. The moving fiber 15 similarly results in lateral movement of the beam at O, which results in the light moving across a sensor array that may extend transversely to the beam, for example, as shown in FIG. 1.

[0024] Figure 3 shows an exemplary fabrication method for creating a support structure to be combined with an array of cell culture units. The fibers are typically very small and very thin, and can be placed over relatively short distances, e.g., 0.1-10 mm. The fiber length is typically in the range of 0.1-0.5 mm, while the diameter is even 10-15 times smaller, with an R / L in the range of 8-15 (see Figure 4).

[0025] In the illustrated example, the fiber diameter ranges from 10 to 50 micrometers. The fiber 15 is fabricated by first selectively etching a shielding layer 20 provided on a glass plate or transparent release layer. The layer thickness can be anywhere from 1 to 100 microns, allowing the corresponding through-hole pattern to be etched. In this example, the shielding layer is a MoCr layer, but other suitable layers are known to those skilled in the art. Accordingly, the support plate is provided with a light-shielding layer to allow only light to pass through the optical fiber. A flowable transparent resin is provided on top of the shielding layer with an appropriate thickness depending on the length of the fiber. The entire structure (excluding the shielding layer 20) is optically transparent, but the top of the pillar 15 can be made reflective by adding an appropriate top layer 22 on the fiber so that the reflected signal from the top of the pillar can be detected in reflection (see Figure D). In this configuration, preferably, light collimated at a certain angle can provide better accuracy. Another advantage of this configuration is that the light source and optical readout device can be installed at the top or bottom of the pillar structure. Finally (Figure E), a clad fiber is produced by cladding with a low refractive index layer (relative to the fiber core) to provide optimal light guidance within the fiber. Depending on the nature of the low refractive index layer, an additional biocompatible layer can be added on top. Alternatively, the optical element 15 can include a light blocking element provided within the elastic support structure, whereby light is at least partially blocked by the light blocking element, resulting in a sensed alignment of the traveling light.

[0026] Instead of a reflective top layer, a refractive optical element can be provided, such as a focusing lens of the type described in Henry E. Williams, et al., "Fabrication of three-dimensional micro-photonic structures on the tip of optical fibers using SU-8," Optics Express, Vol. 19, Issue 23, pp. 22910-22922, (2011) (see FIG. 3). In this way, a refractive element 23 can be provided to refract light to form a light beam that is directed toward the light sensor. Spot sizes even smaller than typical pixel sizes can be obtained. For example, assuming a spot size of 10 microns, the light intensity signal can be easily tracked using an array of light-absorbing (or reflecting) materials patterned on the OPD. The linewidth and spacing of such absorbers can be the same size as the diameter of the light spot (e.g., 10 microns). Thin layers (on the order of 100 nm) can already serve the purpose.

[0027] When the optical fiber is bent due to forces exerted by the tissue (or by flow), the light spot moves and partially hits the absorption pattern as further disclosed below, which effectively modulates the detected intensity and allows for the detection of small displacements at very high readout speeds, since only one pixel per device is required.

[0028] FIG. 5 shows an exemplary deflection curve for a fiber-structured elastic pillar 15, such as those previously disclosed. In another embodiment, hollow pillars can be implemented, with the reflective layer intentionally left exposed at the center of the pillar. This can act as a mirror when backlighting is used, reflecting light back onto the OPD, thereby increasing sensitivity. When viewing the hollow pillar as a collimator, using a mirror on one side makes the collimator appear twice as long, for example, increasing the aspect ratio by a factor of two, depending on the pillar dimensions. Similar deflections of the pillar result in a larger change in signal amplitude.

number

[0029] where E is Young's modulus, R is the radius of the circular pillar, L is the height of the pillar, the point at which the cells / tissues attach, and δ is the displacement. Suitable pillar materials can be, for example, PDMS, TMMF S2000 and TMMR S2000, or PMMA, with Young's moduli ranging from 0.57 to 3.7 MPa (PDMS), 2.1 to 3.8 GPa (TMMF / TMMR), and 2.9 GPa (PMMA).

[0030] The corresponding force / displacement diagrams for different L / R values and different displacements for a Young's modulus of 0.57 MPa are shown, e.g., displacements of 20 microns and L / R values of approximately 20-50 are possible for PDMS pillars for forces as low as 1E-6 Newtons.

[0031] The above calculation relates displacement to force exerted on pillars of different aspect ratios. If a force of 10-6 N is measured, it should be possible to optically detect a displacement of 1 micron, in which case an aspect ratio of about 15 would be sufficient, or alternatively, it should be possible to optically detect a displacement of 10 microns, in which case an aspect ratio of 30 would be required.

[0032] Typical force values range from 10-12 N to 1 N, on the order of magnitude of single cell tissue.

[0033] Figure 6 shows an example spot displacement of an elastic pillar. Furthermore, an estimate of the "amplification" provided by the micromirror configuration is provided below. Assuming a 0.2-degree tilt of the micromirror when a cell / tissue exerts a force, the light spot displacement d projected onto an OPD array positioned at a distance h can be calculated. For example, the displacement (d) can be approximated by tan θ = d / h, where h is the distance between the mirror and the detector. An angle as small as 0.2° can lead to measurable spot displacements. For a pixel size of approximately 100 μm, a displacement of 10 μm is expected to be measurable with a sufficient signal-to-noise ratio. Therefore, in the configuration of Figure 3, for example, assuming a 1-micron displacement can cause a 0.2° tilt, forces of up to 10-100 nN can be accessible by projecting light onto an OPD array at a distance of 1,000 microns.

[0034] FIG. 7 shows several support structures with optical elements, where deformation of the elastic support structure causes light to move over an optical sensor to align the contractile forces of contractile tissue.

[0035] In Figure 7A, no force is applied to the micropillar, causing the light spot (circle) to be centered at a known location (square). When a lateral force is applied, light passing through the micropillar leaves the light at an angle (Figure 7B). In Figures 7C and 7D, such micromechanical movement can be detected with high precision (up to 5 microns) by having light under an angle and casting a shadow of the pillar structure onto the pixel array. In a corresponding way, in Figure 7E, optical shadowing can be used to have light moving above the optical sensor to align the contractile force of contractile tissue. Thus, the micropillar 15 can also reflect or absorb light, thereby altering the non-illuminated pixels (Figure 7F). By having illumination at a shallow angle, the micromovement of the suspended structure is "amplified" across the shadow / light projection.

[0036] In Figure 7G, the entire structure is optically transparent and the tops of the pillars are reflective, allowing the reflected signal from the tops of the pillars to be detected in reflection. In this configuration, preferably, collimated light at an angle can provide better accuracy. Another advantage is that in this configuration, the light source and optical readout device can be placed at the top or bottom of the pillar structure.

[0037] In the presence of contractile tissue exerting a force on the elastic support structure, the deflection of the light can be aligned by the sensors 4 on the sensor plate 7 .

[0038] Optical readers can be used to perform measurements noninvasively, for example, on biomaterials, thereby enabling in vitro analysis of their vitality. They offer high-throughput and parallelized imaging (e.g., acquisition of a complete well plate at high frame rates) without the need for bulky equipment. They can be integrated into well-plate formats or considered as a freestanding platform for optical monitoring of micromechanical (bio)molecular movements. Using light, current / voltage does not directly contact the biological sample, limiting the risk of affecting the measurement. This allows for parallel recording / measurement of micromechanical movements from multiple organs or the same organ interacting with different drugs over long periods of time (days) at high frame rates. This solution can be miniaturized so that the well plate can remain in the incubator (eliminating the need for offline measurements, which potentially disrupt and degrade the quality of the cell culture). In addition to being a generalized, high-resolution imaging device applicable to all types of measurement configurations, imaging plates can be customized to match the specified measurement setup. For example, by confining a high-resolution array of photosensitive pixels to only the center locations of the wells in a smart-well plate, the total number of pixels in the array can be reduced by a large factor, and the frame rate, which is determined by the speed of the readout IC and the number of lines in the imager, can be increased by the same factor. Also, the photosensitive pixel area can be made smaller than the pixel pitch, which allows measurements in reflectance mode.

[0039] In the case of test plates, recording of such micro-movements is only required in a predetermined zone of a large area device, and therefore a series of structures are disclosed to enhance the detection of small movements using a limited number of pixels present in said predetermined region. In this way, high fps can be achieved, as well as high resolution over a large area.

[0040] Although the invention has been described based on preferred embodiments, different embodiments can be used that fall within the scope of the claims, for example, a light guide can be used to guide the light from the light receiving area to a processing unit, which can be arranged outside the incubator.

[0041] Such variations are understood to be within the scope of the present invention as defined in the following claims.

Claims

1. 1. An optical reading device comprising: a cell culture unit capable of culturing contractile tissue; the cell culture unit being provided with an elastic tissue support structure; the elastic tissue support structure extending into the cell culture unit such that the contractile tissue exerts a contractile force on the elastic support structure to generate a deformation of the elastic support structure; the reading device further comprising: one or more light generators and light sensors formed as or including optical elements for deflecting light from the light generator by the elastic support structure onto the one or more light sensors such that deformation of the elastic support structure causes the light to move above the light sensors; and a controller for aligning the movement of the light above the light sensors to measure the contractile force of the contractile tissue.

2. 2. The optical reading device according to claim 1, characterized in that the optical element is designed as an optical fiber.

3. 3. An optical reading device according to claim 1 or 2, characterized in that the optical element is a refractive or reflective element, and light is refracted or reflected to form a light beam that is directed towards the light sensor.

4. 4. The optical reading device of claim 1, wherein the elastic support structure is provided on a support plate that is separated from the test plate including the cell culture unit and is stacked on the test plate such that the elastic support structure protrudes into the cell culture unit of the test plate.

5. The optical reading device of claim 4 , wherein the support plate comprises through-holes for receiving liquid from a well plate to provide culture fluid to the cell culture unit.

6. 6. An optical reading device according to claim 4 or 5, wherein the light generator is provided on the support plate.

7. The optical reading device according to any one of claims 4 to 6, wherein the support plate comprises optical fibers extending outside the cell culture unit.

8. The optical reading device according to claim 7 , wherein the support plate has a light-shielding layer on a surface of the support plate for blocking external light.

9. 9. The optical reading device of claim 1, wherein the optical element comprises a light blocking element provided within the elastic support structure, and wherein light is at least partially blocked by the light blocking element to provide sensed alignment of the moving light.

10. 10. The optical reading device according to any one of claims 1 to 9, characterized in that the housing comprises a base plate in which an optical sensor is provided, said base plate having a shape such that it can be coupled to a test plate containing said cell culture units to provide optical contact.

11. Optical reading device according to any one of the preceding claims, wherein the optical sensor is pixelated.

12. 12. The optical reading device of claim 11, wherein the optical element forms a sub-pixel sized light beam such that a moving spot modulates a single pixel signal.

13. Optical reading device according to any one of the preceding claims, wherein the light sensor is formed by a pixelated organic photodetector.

14. An optical reading device according to any preceding claim, wherein the resilient support structure comprises electrical conductors for providing electrodes for stimulating the contractile tissue.

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