Optical reading device comprising cell culture unit capable of culturing contracted tissue

Abstract

According to one aspect of the present invention, there is provided an optical reading device comprising: a cell culture unit in which contractile tissue can be cultured, in which the cell culture unit is provided with an elastic tissue support structure extending into the cell culture unit such that the contractile tissue applies a contractile force to the elastic support structure, therefore, deformation of the elastic supporting structure is generated; the reading device further comprises one or more light generators and light sensors, where the resilient support structure is formed as or comprises an optical element to transfer light from the light generators onto the one or more light sensors through the resilient support structure such that deformation of the resilient support structure causes the light to move on the light sensors; and a controller for recording the movement of the light on the light sensor, thereby measuring the contractile force of the contractile tissue.

Description

Technical Field

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

[0002] Specifically, the present invention relates to an optical reading device, which includes a housing for receiving a test plate, the test plate providing a cell culture unit according to a fixed pattern, in which a contractile tissue can be formed. The contractile tissue can float in the culture unit. In the prior art, the cell culture unit is provided with an elastic support structure extending into the cell culture unit so that the contractile tissue exerts a contractile force on the elastic support structure, thereby causing deformation of the elastic support structure.

[0003] One of the challenges with this system is the difficulty in generating a large-scale testing environment, as the system requires detailed optical studies of the mechanical interactions between the tissue and the supporting structure, for example by using imaging, in particular video confocal microscopy. This is because in typical cases the contraction may not exceed a few micrometers, which is difficult to study. Therefore, the structure is imaged and the images need to be studied. This imaging is costly and labor intensive. As a result, current solutions have low throughput, provide only snapshots, and / or may negatively impact drug testing.

[0004] It is an object of the present invention to provide an optical reading device which can be easily produced on an industrial scale and at competitive costs. Summary of the invention

[0005] According to the present invention, this object and these aims as well as other objects which will be apparent from the following description and the accompanying drawings are achieved by a reflective structure according to claim 1 as set forth below.

[0006] An optical reading device includes a cell culture unit in which contractile tissue can be cultured. The cell culture unit is provided with an elastic tissue support structure, which extends into the cell culture unit so that the contractile tissue exerts a contractile force on the elastic support structure, thereby generating a deformation of the elastic support structure. The reading device also includes one or more light generators and light sensors. The elastic support structure is formed as an optical element or includes an optical element so that light is transferred from the light generator to one or more light sensors through the elastic support structure. In this way, the deformation of the elastic support structure causes the light to move on the light sensor. A controller is used to record the movement of light on the light sensor, thereby measuring the contractile force of the contractile tissue.

[0007] Thanks to the invention, an optical reading device can be provided in which the test can be performed without being disturbed by the measurement. It is worth noting that an elastic support structure of the above-mentioned type is itself known from WO2021206551. However, this is a system that is not suitable for imaging large areas.

[0008] According to the described imaging system, an optical reading device can be provided, in which mechanical movements can be efficiently and easily monitored on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Further features and advantages of the elastic support structure of the present invention will become more apparent with reference to the following description and accompanying drawings, which are provided for illustrative purposes only and not for limiting purposes, in which:

[0010] Figure 1 An embodiment of an optical reading device is shown (exploded view), along with a schematic cell culture unit array;

[0011] Figure 2 An alternative embodiment of a resilient support structure formed from optical fibers is shown;

[0012] Figure 3 (AE) illustrate an exemplary fabrication method for creating a support structure to be coupled to an array of cell culture units;

[0013] Figure 4 A support structure with a focusing lens is shown;

[0014] Figure 5 An exemplary deflection curve for an elastic column is shown;

[0015] Figure 6 An exemplary spot displacement of an elastic column is shown;

[0016] Figure 7 (AG) show multiple support structures with optical elements. DETAILED DESCRIPTION

[0017] Hereinafter, exemplary embodiments of the present invention will be described with reference to the accompanying drawings.

[0018] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as those generally understood by a person of ordinary skill in the art to which the present invention belongs in the context of the specification and the accompanying drawings. It should also be understood that terms (such as those defined in commonly used dictionaries) should be interpreted as having a meaning consistent with the meaning in the context of the relevant field, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein. In some cases, a detailed description of well-known devices and methods may be omitted so as not to obscure the description of the present system and method. The terms used to describe a particular embodiment are not intended to limit the present invention. As used herein, the singular forms "one", "an" and "the" are also intended to include plural forms unless the context clearly indicates otherwise. The term "and / or" includes any and all combinations of one or more related listed items. It should also be understood that the terms "include" and / or "comprising" specify the presence of the features described, 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 the event of a conflict, this specification (including definitions) shall prevail.

[0019] The optical reader can be provided as a large area optical imager, either as an integrated device or as a stand-alone device, for measuring micro (bio) motions and deriving mechanical parameters such as flow and / or contraction forces (e.g. quantitative measurements during drug screening). In one embodiment, the mechanically flexible micropillars act as optical fibers, directing light onto a pixel (or pixel array), and the deflection of such optical fibers under external stimuli is converted into light intensity changes or spot displacements. It should be noted that high positional accuracy (better than pixel pitch) can be obtained by interpolating using intensity and spatial information.

[0020] Although example embodiments are shown for the systems and methods, those skilled in the art, with the benefit of this disclosure, may also envision alternative methods to achieve similar functions and results. For example, some components may be combined or split into one or more alternative components. Finally, these embodiments are intended only to illustrate the present system and should not be construed as limiting the appended claims to any particular embodiment or group of embodiments. Therefore, although the present system has been described in particular detail with reference to its specific exemplary embodiments, it should also be understood that a person of ordinary skill in the art may design many modifications and alternative embodiments without departing from the scope of the present system and method as described in the following claims. Therefore, the description and drawings should be regarded in an illustrative manner and are not intended to limit the scope of the appended claims.

[0021] Preferably, the size of the optical reading device is only slightly larger than a standard culture plate so that the reading device can be contained in an incubator and read in the incubator. Thus, real-time measurements can be performed without changing the conditions of the cell culture. Due to this configuration, the reader can be designed to be compact and can be coupled in a planar manner so that it can be placed in an incubator as a whole.

[0022] Although different light guide structures can be envisaged, a convenient form of combining the elastic structure and the light guide is provided by an optical element designed as an optical fiber. The optical element can be a refractive or reflective element so that the light is refracted or reflected to form a beam directed to the light sensor. This enlarges the physical deflection of the structure to a certain extent, so that it can be measured more easily. The elastic support structure can be arranged on a support plate separated from the test plate and stacked thereon, so that the elastic support structure protrudes into the cell culture unit of the test plate. Having a separate support plate is a convenient way to provide an optically optimized structure that is also suitable for contractile tissue to be grown and the optical structure is arranged in the culture unit. For example, the support plate can be additionally provided with a through hole for receiving a fluid from the well plate so as to provide a culture fluid for the cell culture unit. In addition, a light generator can be arranged on the support plate. In addition, an optical fiber arranged in a pattern corresponding to the cell culture unit can be provided on the support plate. Alternatively, the support plate is provided with a light shielding layer on the surface of the support plate to allow only light to pass through the optical fiber. For example, the optical element can be a light shielding element arranged in the elastic support structure so that the light is at least partially shielded by the light shielding element, resulting in a sensing record of the moving light. In another embodiment, the housing comprises a substrate in which the light sensor is provided, the substrate having a shape that can be coupled to the test board to provide optical contact. In another example, the light sensor is pixelated. In some embodiments, the optical element forms a light beam of sub-pixel size so that the moving light spot modulates a single pixel signal. This provides a very fast readout of small deviations without much processing load and is therefore suitable for further efficient scaling. In an embodiment, the light sensor is formed by a pixelated organic photodetector. The elastic support structure can also be provided with electrical conductors for providing electrodes to stimulate the contracting tissue.

[0023] In one embodiment, the resilient support structures may be spaced apart a distance between 0.1 and 10 mm, preferably between 0.1 and 5 mm, preferably between 0.1 and 3 mm, preferably between 0.1 and 2 mm, preferably between 0.1 and 1 mm, for example about 0.6 mm or about 0.9 mm.

[0024] Further references Figure 1, an embodiment of an optical reading device 1 is shown, in which a schematic cell culture unit array 6 is also shown in an exploded view. The optical reader 1 comprises a housing 2 with a planar test plate 3, on which the cell culture units 6 are arranged. The cell culture units 6 of the test plate 3 form a fixed pattern, in the example in the figure a matrix-like pattern, which is composed of a large number of wells, which is common in practice, such as a 96-well plate. In the figures, for the sake of clarity, only a limited number of wells are shown. On another sensor component 7, the optical conversion element 4 is, for example, a photodiode, or a different type of semiconductor that is sensitive to the relevant optical range.

[0025] The conversion elements 4 are arranged in the sensor part 7 in a pattern similar to the holes 6 of the test plate 3. The dimensions of the device according to the invention correspond in practice to the dimensions of a standard test plate, so that the reading device can be contained in an incubator (not shown) for the cultivation of biological material and can be read in situ without mechanical disturbances, in particular without removing the plate from the incubator. To this end, the invention comprises a method for testing contractile tissue using an optical reader, the method comprising the steps of providing the contractile tissue in a test plate 3 comprising a cell culture unit 6, coupling the reader to the test plate and inserting the reader into the incubator, while the measurement signals from the reader are stored in a memory of the reader and / or output to a central processing unit.

[0026] On the test plate 3, the coupling element 8 may be present in the form of a groove in which the test plate can be slid and fixed to form a fixed connection with the optical reader 1. In addition, an elastic support structure 15 is provided on a support plate 5 that is separate from the test plate 3 including the cell culture unit 6 and is stacked thereon. In this way, the elastic support structure 15 protrudes into the cell culture unit 6 of the test plate 3. The elastic support structure 15 is suitable for supporting a contractile tissue so that the tissue can exert a contractile force on the support structure to deflect it, as described below. To this end, the support structure 15 includes an optical element, which may have a shape such as Figure 5 Different embodiments shown. In the embodiment shown, the optical element is designed as an optical fiber adapted to provide support for the tissue, for example by having a biocompatible cladding. Thus, the support plate 5 is provided with optical fibers 15 arranged in a pattern corresponding to the cell culture units 6.

[0027] Via connections 9, the optical converters 4, for example formed by pixelated organic photodetectors, are connected to an electronic control unit 10 for controlling the individual converters 4, in particular for controlling the timing of reading the signals from the converters. By means of external couplings, the control unit 10 can be connected to a computer as a peripheral device.

[0028] The reader 1 is adapted to analyze light from a well 6 in a test plate 3. To this end, in the optical reader 1, a light generator 13 is provided on the support plate 5, but coupling can also be performed via other optical coupling structures. In the embodiment shown, the light source part support plate 5 comprises one or more light generators and light sensors, and an elastic support structure 15 formed as an optical element, so that one or more light beams generated by the light generator 13 are redirected to the light sensor of the sensor part 7 by the elastic support structure. The reader may also include additional structures, such as a well plate 11. In the structure shown, the support plate 5 is also provided with through holes for receiving fluid from the well plate, so as to provide a culture fluid to the cell culture unit.

[0029] Figure 2 An alternative embodiment of a resilient support structure formed as an optical fiber is shown. In variant A, the optical fiber 15 is part of a support plate 5 or other support structure. Tissue attached to the optical fiber 15 exerts a contraction force F thereon, for example by attachment to a portion of a hole or other support element (not shown). Light I is injected into the optical fiber, producing a moving light beam O that can be detected as it moves across a light sensor, which can detect lateral movement of the light beam O. In variant B, the optical fiber can be part of a cell culture unit 6, for example, protruding through a bottom wall. The contraction force F causes movement of the optical fiber into which the light is injected at I. Moving the optical fiber 15 will similarly cause the light beam to move laterally at O, which causes the light to move across an array of sensors that can extend laterally to the light beam, for example as Figure 1 shown.

[0030] Figure 3 An exemplary fabrication method for creating a support structure to be coupled to an array of cell culture cells is shown. The fibers are typically very small and very thin, and can be placed at relatively short distances, for example between 0.1 and 10 mm. The fiber length is typically in the range of 0.1 to 0.5 mm, while the diameter is even 10-15 times smaller, for R / L (see Figure 4 ), ranging from 8 to 15.

[0031] In the illustrated example, the fiber diameter ranges between 10 and 50 microns. The optical fiber 15 is produced by first selectively etching a shielding layer 20 on a glass plate or on a transparent release layer. The layer thickness can be any value between 1 and 100 microns so that a pattern of corresponding through holes can be etched. In the example, the shielding layer is a MoCr layer, but other suitable layers are known to those skilled in the art. Therefore, the support plate is provided with a light shielding layer to allow only light to pass through the optical fiber. A fluid transparent resin is provided on the shielding layer, and its thickness corresponds to the length of the optical fiber. The entire structure (except the shielding layer 20) is optically transparent, and the top of the column 15 can be made reflective by adding a suitable top layer 22 on the optical fiber (see Figure D), so that the reflected signal from the top of the column can be detected in reflection. In this configuration, preferably, collimated light at a certain angle can achieve better accuracy. Another advantage is that with this configuration, the light source and the optical readout device can be placed at the top or bottom of the column structure. Finally (Figure E) the optical fiber is coated with a low refractive index layer (relative to the optical fiber core) to produce a coated optical fiber to provide optimal light guidance in the optical fiber. Additional biocompatible layers may be added on top depending on the properties of the low refractive index layer.Alternatively, the optical element 15 may comprise a light shielding element arranged in an elastic support structure such that light is at least partially shielded by the light shielding element, resulting in a sensed registration of moving light.

[0032] An alternative to a reflective top layer is a refractive optical element, such as a focusing lens as described by Henry E. Williams et al. in "Fabrication of Three-Dimensional Microphotonic Structures at the Tip of Optical Fibers Using SU-8", Optics Express, Vol. 19, No. 23, pp. 22910-22922 (2011), see Figure 3 . In this way, a refractive element 23 can be provided so that the light is refracted to form a light beam directed to the light sensor. Spot sizes 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) material patterned on top of the OPD. The line width and spacing of such absorbers can be the same as the spot diameter (e.g. 10 microns). Thin layers (about 100 nanometers) can already achieve this purpose.

[0033] When the fiber bends due to forces exerted by the tissue (or flow), the light spot moves and partially hits the absorption pattern, as described below. This effectively modulates the detected intensity, enabling the detection of small displacements at very high readout speeds, since only one pixel is required per device.

[0034] Figure 5An exemplary deflection curve is shown for an elastic post 15, for example, a fiber structured elastic post as previously described. In another embodiment, a hollow post can be implemented with the reflective layer intentionally exposed in the center of the post. When backlighting is used, this can act as a mirror so that light is reflected back to the OPD, thereby increasing sensitivity. Considering the hollow post as a collimator, using a mirror on one side will make the collimator appear to have a 2x aspect ratio and double the length, depending on the post size. Similar deflection of the post will result in a larger change in signal amplitude.

[0035]

[0036] Where E is Young's modulus, R is the radius of the cylinder, L is the height of the cylinder, i.e. the point of cell / tissue attachment, and δ is the displacement. Suitable cylinder materials can be PDMS, TMMF S2000 and TMMR S2000, or PMMA, with Young's modulus ranging from 0.57-3.7 MPa (PDMS); 2.1-3.8 GPa (TMMF / TMMR); and 2.9 GPa (PMMA).

[0037] The figure shows the corresponding force / displacement graphs for different L / R values ​​and different displacements when the Young's modulus is 0.57 MPa. For example, for PDMS pillars, the displacement is 20 microns at a force as low as 1E-6 Newton, and the L / R value is about 20-50.

[0038] The above calculation relates displacement to force applied to a post of different aspect ratios. If you want to measure a force of 10-6N, you should be able to optically detect a displacement of 1 micron, in which case an aspect ratio of 15 is sufficient, or a displacement of 10 microns, in which case an aspect ratio of 30 is required.

[0039] Typical force values ​​range from 10-12N to 1N for single cell tissue.

[0040] Figure 6 An exemplary spot displacement for an elastic post is shown. Additionally, an estimate of the "magnification" provided by the micromirror configuration is provided below. Assuming the micromirror is tilted 0.2 degrees when a cell / tissue applies force, the spot displacement d projected onto the OPD array at a distance h can be calculated. For example, the displacement (d) can be approximated by tan 2θ = d / h, where h is the distance between the mirror and the detector. An angle as small as 0.2 degrees can result in a measurable spot displacement. For a pixel size of 100 μm, a displacement of 10 μm is expected to be measurable with adequate signal-to-noise ratio. Therefore, for Figure 3 The configuration, for example, assumes that a 1 micron displacement results in a 0.2 degree tilt, and that the force can be reduced to 10-100 nN by projecting light onto the OPD array 1000 microns away.

[0041] Figure 7Multiple support structures with optical elements are shown, where deformation of the elastic support structure causes light to move on a light sensor to record the contractile force of the contracting tissue.

[0042] exist Figure 7 In A, no force is applied to the micropillar, which results in a light spot (circle) centered at a known position (square). When a lateral force is applied, light passing through the micropillar exits at an angle ( Figure 7 B) In Figure 7 C and Figure 7 D, this micromechanical movement can be detected with high precision (down to 5 microns) by placing light at a certain angle and projecting the shadow of the pillar structure onto the pixel array. Figure 7 In E, light shadowing can be used to move light across the light sensor to record the contractile force of the contracting tissue. Therefore, micropillars 15 can also reflect or absorb light, causing the unilluminated pixels to change ( Figure 7 F). By lighting at shallow angles, micro-movements of the suspended structure are “magnified” in the shadow / light projection.

[0043] exist Figure 7 In G, the entire structure is optically transparent and the top of the pillar is reflective, so the reflected signal from the top of the pillar can be detected from the reflection. In this configuration, collimated light, preferably at a certain angle, can improve accuracy. Another advantage is that with this configuration, the light source and optical readout device can be placed at the top or bottom of the pillar structure.

[0044] When the contracting tissue exerts a force on the elastic support structure, the sensors 4 on the sensor plate 7 can register light deflections.

[0045] Using an optical reader, measurements of biological materials can be performed in a non-invasive manner, for example, their viability can be analyzed in vitro. It provides high throughput and parallel images (e.g., acquisition of a complete well plate at high frame rates) without the need for bulky instruments. It can be integrated into a well plate format or designed as a stand-alone platform for optical monitoring of micro(bio)mechanical motion. When using light, the current / voltage does not directly contact the biological sample, limiting the risk of influencing the measurement. It enables parallel recording / measurement of micromechanical motion of multiple organs or interactions with different drugs over long periods of time (days) at high fps. The solution can be miniaturized, so the well plate can remain in the incubator (without offline measurements, which can interfere with and reduce the quality of the cell culture). In addition to universal high-resolution imagers for various measurement configurations, imaging plates can also be custom designed to match a specified measurement setup. For example, by limiting the high-resolution array of photosensitive pixels to near the center of the well in a smart well plate, the total number of pixels within the array can be greatly reduced, while the frame rate can be increased by the same factor, as it is determined by the speed of the readout IC and the number of rows in the imager. In addition, the photosensitive pixel area can be smaller than the pixel pitch. This allows measurements to be made in reflection mode.

[0046] Since in the case of the test plate it is only necessary to record such micro-movements in a predetermined area of ​​a large-area device, a series of structures are disclosed with the aim of enhancing the detection of tiny movements using the limited number of pixels present in the above-mentioned predetermined area. In this way, high fps as well as high resolution over a large area can be achieved.

[0047] Although the present invention has been described based on preferred embodiments, different embodiments are also possible and fall within the scope of the claims. For example, a light conductor can be used to conduct light from the light receiving area to a processing unit, which can even be arranged outside the incubator or elsewhere.

[0048] It is understood that such modifications fall within the scope of the present invention as defined by the following claims.

Claims

1. An optical reading device, comprising: A cell culture unit in which contractile tissue can be cultured, wherein the cell culture unit is provided with an elastic tissue support structure, the elastic tissue support structure extending into the cell culture unit so that the contractile tissue applies a contractile force to the elastic support structure, thereby generating a deformation of the elastic support structure, the reading device further comprising one or more light generators and light sensors, wherein the elastic support structure is formed as an optical element or comprises an optical element so as to transfer light from the light generator to the one or more light sensors through the elastic support structure so that deformation of the elastic support structure causes light to move on the light sensors, and a controller for recording the movement of light on the light sensors, thereby measuring the contractile force of the contractile tissue.

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

3. An optical reading device according to any one of the preceding claims, characterized in that The optical element is a refractive element or a reflective element, and light is refracted or reflected to form a light beam directed toward the light sensor.

4. An optical reading device according to any one of the preceding claims, characterized in that The elastic support structure is disposed on a support plate separated from a 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 according to claim 4, characterized in that The support plate is provided with through holes for receiving fluid from the well plate so as to provide culture fluid to the cell culture unit.

6. The optical reading device according to claim 4 or 5, characterized in that: The light generator is disposed on the supporting plate.

7. The optical reading device according to any one of claims 4 to 6, characterized in that: The support plate is provided with an optical fiber extending to the outside of the cell culture unit.

8. The optical reading device according to claim 7, characterized in that A light shielding layer is arranged on the surface of the support plate to block ambient light.

9. An optical reading device according to any one of the preceding claims, characterized in that The optical element includes a light shielding element disposed in the elastic support structure, and the light shielding element at least partially shields light, thereby resulting in a sensed registration of moving light.

10. An optical reading device according to any one of the preceding claims, characterized in that The housing includes a substrate in which a light sensor is disposed, the substrate having a shape capable of being coupled to a test plate including the cell culture unit to provide optical contact.

11. An optical reading device according to any one of the preceding claims, characterized in that The light sensor is pixelated.

12. The optical reading device according to claim 11, characterized in that The optical element forms a sub-pixel sized light beam so that the moving light spot modulates a single pixel signal.

13. An optical reading device according to any one of the preceding claims, characterized in that The light sensor is formed by pixelated organic photodetectors.

14. An optical reading device according to any one of the preceding claims, characterized in that The resilient support structure is provided with electrical conductors for providing electrodes for stimulating the contracting tissue.

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