Claims:We claim
1. A microscope mountable shear device (A), comprising:
Cone plate-motor alignment stage (I) attached to translation stage (II), wherein cone plate- motor alignment stage (I) further comprises
a motor (2) coupling perpendicular to the cone plate (1) and precision alignment screws for tilt correction (3);
translation stage (II) further comprising an alignment stage (4) and base plate (8) with an aperture; and
transparent bioreactor stage (III) mounted on the base plate (8) of translation stage (II),wherein the translation stage (II) and bioreactor stage (III) are fixed through sliding joint arrangement (5).
2. The microscope mountable shear device (A) as claimed in claim 1, wherein the cone plate (1) is of cone angle ß ranging from about 1° to less than 5°.
3. The microscope mountable shear device (A) as claimed in claim 1, wherein the cone is of a material selected from a group comprising aluminium and stainless steel; preferably aluminium.
4. The microscope mountable shear device (A) as claimed in claim 1, wherein the bioreactor stage (III) comprises carbon dioxide to maintain cell viability.
5. The microscope mountable shear device (A) as claimed in claim 1, wherein the bioreactor stage (III) comprise temperature controllers for maintaining temperature inside bioreactor stage (III).
6. The microscope mountable shear device (A) as claimed in claim 5, wherein the temperature is ranging from about 25°C to about 40°C.
7. The microscope mountable shear device (A) as claimed in claim 1, wherein the device (A) is mounted on an inverted microscope.
8. A method of measuring adhesion of micro patterned surface bound objects using device (A) of claim 1, comprising acts of
a) growing or adhering the object on a micro-patterned substrate in a container;
b) placing the container on aperture of the device (A) in transparent bioreactor stage (III);
c) calibrating the device to maintain a distance ranging from about 10µm to about 30µm between tip of cone and the object;
d) rotating cone ranging between 90 rpm and 800rpm to apply shear stress; and
e) measuring the adhesion by quantifying the number of objects adhering on the substrate.
9. The method of measuring adhesion of micro patterned surface bound objects as claimed in claim 8, wherein the substrate is a transparent substrate.
10. The method of measuring adhesion of micro patterned surface bound objects as claimed in claim 8, wherein surface bound object is selected from a group comprising cells, biomolecules, and soft matter.
11. The method for measuring the adhesion of micro patterned surface bound objects as claimed in claim 10, wherein the surface bound objects are cells.
12. A method of distinguishing healthy cells from cancer cells, said method comprising act of measuring the adhesion of cells on micropatterned substrates using microscope mountable shear device (A) of claim 1.
13. A substrate comprising micro-patterns for constraining objects and measuring adhesion of the object to surface of the substrate.
14. The substrate as claimed in claim 13, wherein the substrate is selected from a group comprising poly dimethyl siloxane and polyacrylamide.
, Description:Technical Field
The present invention relates to the field of fluid dynamics, complex fluids and biophysics. More specifically, the invention is in relation to a device designed for generating shear stress for various analytical studies. In particular, a device designed for generating shear stress on biological materials, soft matter and the like for analytical studies.

Background
Shear stress is the force per unit area that is generated on a surface due to fluid flow over the surface. This force generated is parallel to the surface. Some cells in our body experience fluid shear stresses. For example, the endothelial cells lining the blood vessels and the epithelial cells lining the renal tubules are constantly exposed to shear stress due to the flow of fluids over their surface. These cells respond to stress by changes in morphology, cell signalling and gene expression. A device which can generate shear stress is needed to study the effect of the shear forces on cell morphology in a controlled environment. Application of controlled shear stress can also be used as an effective tool to study other cell properties like adhesion and bio-mechanical responses which may alter under disease conditions.
The effect of shear stress has been analysed by applying stress on cells using various tools, commonly used tools which include cell monolayer rheometer, ?ow chambers, and the like. The cell monolayer rheometer utilizes a commercial rheometer with some modifications to apply shear stress and is capable of applying a constant or cyclic shear stress or strain to study the cell properties. The shear stress can be applied by sliding one plate over the other in linear fashion or in a cyclic manner. However, the stress cannot be applied constantly through extended periods. Also, the device being bulky cannot be combined with a microscope for a magnified image of the cell layer under study. Commercial rheometers are also very expensive. Similarly, flow chambers are compact, low cost, and easy to handle tools and can be used with different types of microscopes, but it requires huge amount of medium if the shear stress analysis is to be carried for hours and thereby requiring added machinery for supplementing the media. Some of these tools do not permit the ability to change the shear stress easily without disturbing the setup and cannot be used for extended duration.
Current commercial devices are very expensive and bulky, and added to the base cost of the instruments are the excess costs in consumables for each experiment which include custom channels, clamps, fixtures, and large amount of media for biological applications. Some of these devices are constructed to characterize rheological properties of complex fluids also and do not readily offer the option to visualize cells real time that warrant purchase of special microscope and camera setup which adds to the operating costs of the setup.
Patent application US 20160024454 explains a device to expose cells to fluid shear forces. The device includes a flow unit configured to induce fluid flow through the device. The device also includes a fluid channel configured to accept a biological sample dispersed on an array of flexible structures. The flow unit can be configured to induce disturbed and/or laminar flow in the fluid channel.
Patent application US 20080057571 explains a bioreactor device for exposing cell culture to fluid shear force imparted by a fluid flow. The invention provides flow chambers defining channels for retaining a cell culture in the fluid flow wherein the flow chambers may be removably disposed within a bioreactor system such that the flow chambers may be removed and/or replaced without disturbing the cell cultures retained therein or disposed elsewhere within the bioreactor system. The flow chambers are composed of a transparent material such that a user of the system may observe the development of the cell culture retained within the chamber as the cell culture is imposed to a fluid shear stress imparted by the fluid flow.
However the aforementioned devices are bulky and expensive, also they cannot provide a magnified image of the cell culture during the application of stress which is essential in order control the rate of stress and the various parameters during the experimentation process.
Hence there is a need for a compact, low cost, microscope mountable device for high end imaging of specimens under shear stress and which can be adapted for various biological applications by suitably controlling the various parameters like temperature, humidity during the entire course of the experiment.

Summary of invention
Accordingly, the present invention is in relation to a microscope mountable fluid shear device for generating shear stress on biological samples, soft matter and the like for various analytical studies. The fluid shear device comprises of a cone plate- motor alignment stage with translation stage aligned together by a sliding joint; wherein the cone plate stage comprises a motor coupled perpendicular to the cone plate and, precision alignment screws for tilt correction. The translation stage comprises a base plate with an aperture. This stage further comprises a bioreactor stage mounted on the base plate of the translation stage. The device finds use in investigating responses of the live biological materials for mechanical stimuli under controlled environment.

Brief description of figures
The features of the present invention can be understood in detail with the aid of appended figures. It is to be noted however, that the appended figures illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope for the invention.
Figure A: depicts a fluid shear device comprising a cone plate stage(I) , translational stage (II) and bioreactor stage(III).
Figure 1A: shows the exploded view of the device assembly comprising a cone plate(1) powered by a motor (2) coupled perpendicular to the cone-plate arrangement of the device, alignment stage (4)of translation stage (II) aligned together with the base plate (8) by a sliding joint (5) and alignment screws(3). The base plate (8) is provided with a petri-dish with micro-patterned cover slip bottom plate(7). The cone plate (1) and the base plate (8) are together enclosed in bioreactor stage(6). Figure 1B shows the assembled CAD design of the device.
Figure 2: explains the microscope stage to aid mounting of the device on the microscope, the cone plate, motor arrangement and the alignment stage.
Figure 3: shows a prototype of the miniature fluid shear device with microscope stage sans bioreactor chamber to permit easy visualization of the different components of the device.
Figure 4A: depicts the inverted microscope stage to mount the fluid shear device.
Figure 4B: depicts a CAD drawing of the stage insert used to design the shear device prototype base. Figure 4C. shows the coupling plate and the condenser of the inverted microscope which is tilted to allow the shear device to be mounted for experimentation.
Figure 5: explains the working principle of the fluid shear device with a rotating cone placed above a micro-patterned substrate to investigate cellular responses.
Figure 6: Figure 6A shows a bright field image of the cone apex, Figure 6B explains the height difference between the edge of the cone and the apex used to obtain the angle ß.
Figure 7: Figure 7A shows a pseudocolor plot for the surface roughness (Ra) of the cone at the apex, Figure 7B shows surface roughness at locations away from the apex.
Figure 8: Figure 8Ashows scanning electron micrograph of a sample substrate of micro-fabricated array of circular patterns, Figure 8B shows a fibronectin coated stamped cover slip which shows good fidelity in the transfer of the pattern from the micro-fabricated shape
Figure 9: Figure 9a provides a plot to show differences in the de-adhesion dynamics of mouse fibroblasts (3T3) and HEK cells in the shear device. Figure 9b: provides a plot to explain cells treated with blebbistatin showed faster detachment initially as compared to non-treated cells; the detachment becomes slower over longer time duration. The detachment curves are taken as an average over four independent runs.
Figure 10: Figure 10a shows 3T3 cells transfected with vinculin GFP, cultured on micro-patterned coverslip coated with fibronectin, which clearly shows focal adhesions in the cell maintained under static (no shear) conditions. Figure 10b shows change in cell shape and redistribution of the focal adhesion observed after 30 minutes of 1 Pa fluid shear stress.
Figure 11: provides a schematic diagram showing the working principle of the fluid shear device.

Detailed description of invention:
The foregoing description of the embodiments of the invention has been presented for the purpose of illustration. It is not intended to be exhaustive or to limit the invention to the precise form disclosed as many modifications and variations are possible in light of this disclosure for a person skilled in the art in view of the figures, description and claims. It may further be noted that as used herein and in the appended claims, the singular “a” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by person skilled in the art.
The present invention is in relation to a microscope mountable fluid shear device for generating shear stress in biological samples, soft matter and the like for various analytical studies in a controlled environment. The device combines the use of micro-patterned substrates to control cell shape and adhesion.

The present invention is in relation to a microscope mountable shear device (A), comprising:
cone plate-motor alignment stage (I) attached to translation stage (II), wherein cone plate-
motor alignment stage (I) further comprising:
a motor (2) coupling perpendicular to the cone plate (1) and precision alignment screws(3);
translation stage (II) further comprising an alignment stage (4) and base plate (8) with an
aperture; and
transparent bioreactor stage (III) mounted on the micro-patterned base plate (8) of translation stage (II), wherein the translation stage (II) and bioreactor stage (III) are fixed through sliding joint arrangement (5).
In another embodiment of the invention the shear device is powered by a motor coupled perpendicular to the cone plate arrangement of the device.
In another embodiment of the invention, the translational stage comprises an alignment stage and a base plate with an aperture.
In yet another embodiment of the invention, the translational stage comprises a bioreactor stage mounted on the base plate of the translation stage to maintain cell viability.
In another embodiment of the invention, the cone angle of the cone plate ranges from 1° to less than 5°.
In yet another embodiment of the invention, the material of the cone is selected from a group comprising aluminium, and stainless steel; preferably aluminium.
In yet another embodiment of the invention, the bioreactor stage is a CO2 chamber to maintain cell viability.
In another embodiment of the invention, the bioreactor stage is attached with temperature controllers to maintain temperature within the bioreactor.
In another embodiment of the invention, the temperature of the bioreactor stage is ranging from about 25°C to about 40°C preferably about 37±0.5 °C.
The present invention is also in relation to a method for measuring adhesion of micro patterned surface bound objects using the fluid shear device comprising acts of growing or adhering the object on a micro-patterned substrate in a container; placing the container on aperture of the device in transparent bioreactor stage; calibrating the device to maintain a distance ranging from about 10µm to about 30 µm between tip of cone and the object; rotating cone maintaining the angular velocity ranging between 90 rpm and 800rpm to apply shear stress; and measuring the adhesion by quantifying the number of objects adhering on the substrate.
In another embodiment of the invention the micro-patterned substrate is a transparent substrate.
In another embodiment of the invention, the surface bound object is selected from a group comprising cells, biomolecules and soft matter.
The present invention is also in relation to a method of distinguishing healthy cells from cancer cells, said method comprising act of measuring the adhesion of cells on micropatterned substrates using microscope mountable shear device (A).
In another embodiment of the invention, the substrate is micro-patterned to constrain the objects and adhesion.
In another embodiment of the invention, the substrate is selected from a group comprising poly dimethyl siloxane and polyacrylamide.
The present invention is also in relation to a device designed for simulating fluid shear stress on various cell types in natural physiological environment. The device (Figure A) is divided into three parts: first, a cone plate-motor alignment stage (I), base-translation stage (II) and a bioreactor stage (III). The translation stage (II) and bioreactor stage (III) are fixed together through a sliding joint arrangement (5) (HolmarcOpto-Mechatronics; 10 µm resolution). The sliding joint arrangement (5) is used to position the cone above the sample and aligning the orientation of the cone with respect to the base substrate. A hard drive motor (2) coupled perpendicularly to the cone-plate is used to run the device at various speeds using an electronic speed controller to generate the shear force in the fluid. The cone plate motor alignment stage is also provided with precision alignment screws (3) for tilt adjustment. The translation stage (II) comprises an alignment stage (4) and a base plate (8) with an aperture for inserting the petridish with micro patterned substrate(7). This stage further comprises a bioreactor (6) mounted on the base plate (8). The working principle of the device is schematically shown in Figure 11. An IR sensor is used to determine the speed of the cone during rotation which is controlled using software. The cone of the cone plate alignment stage generates a laminar flow and is selected to biological applications. The material of the cone is selected from a group comprising, biocompatible materials such as aluminium and stainless steel alloys; preferably aluminium.
Secondly, it is easy to sterilize for each experiment to maintain sterility. Thirdly, the cone has a good surface finish (figure 6), and is sufficiently light to allow use of the motor to generate chosen torques (figure 7).
The base plate (8) of the translational stage (II) of the device is provided with an aperture for mounting a petridish comprising sample on micro- patterned substrate. The sample may be grown or adhered on the substrate. The substrate is selected from poly dimethyl siloxane, polyacrylamide and other hydrogels which permit cell adhesion.
Micro-patterning of substrates(Figure 8A) is done to obtain consistent results on cell de-adhesion assays and to quantify the roles of shears in cellular morphology. Soft lithography is used to obtain stamps which are created using a replica moulding process in microfabrication. In this method, silicon wafers are coated with SU8, exposed to UV with a mask containing the desired pattern which is placed in the optical path, and cured to obtain a negative master mould of the pattern. Poly dimethyl siloxane (PDMS) is poured on the master mould, cured and used as a positive master which contains an array of the desired features and patterns. The PDMS patterns arecoated uniformly with extracellular matrix proteins (figure 8B) (fibronectin/ lamin/ collagen I) and are placed on piranha cleaned and activated cover slips to stamp the pattern on the cover slip. The use of soft lithography is essential in constraining cells and in adhesion of soft matter within patterned substrate.
A bioreactor (Figure 1B) is mounted on the aperture of base plate (8) for maintaining uniform standards of various parameters during the experimentation. The bioreactor essentially consists of a chamber with four sides, two of which are made of aluminium to permit heating of the wall and the other two of transparent material for ease in visualization. Ports allow inlet/ outlet of CO2 mixture which is essential to maintain the pH of biological cells and tissues. Additional ports are included for addition of biologicals and media into the petridish during experimentation. Temperature controllers are attached to the base plate and the side walls of the aluminum plate, to maintain the temperature of the petridish within a range of about 25°C to about 40°C preferably 37±0.5 °C for cell culture conditions. All components may be dis-assembled and sterilized prior to the start of the experiment.
The fluid shear device is light and compact and is mounted on an inverted microscope (figure 4) for high end imaging of the specimens when subjected to shear stress. The inverted microscope has a stage with insert plates to place biological samples. The outer dimensions of the insert plates are maintained to match the aperture in the base plate of the fluid shear device. This allows the device to be placed on motorized stage of the inverted microscope and also permits multi point imaging via the motorized stage to visualize during experimentation. The aperture in the base plate of the device is used to snugly place a 35/60 mm petridish for live cell imaging
The invention is based on the principle of a cone-plate rheometer. A Couette flow is achieved through rotation of the cone placed above the base plate with a fluid between the two components, the cone plate and the base plate.
The experimentation involves placing the petri plate under the cone, followed by alignment process wherein the cone is brought closer to petri dish using the translation stage and the alignment screws on the stage. The alignment of the cone axis, perpendicular to the cell culture dish, is very crucial as any tilt in the axis would cause different fluid shear at different point. In order to make the rotating axis of the cone perpendicular to the surface of the cell culture, a motorized stage of Axio Observer Z1 microscope along with Axio Vision software, is used. Alignment is done by focusing at the cone surface and the dish surface at four points which are equidistant from the cone centre. The steps adopted to align the cone with respect to the plate are as follows. First, a dish with fluorescent markers is placed on the base plate in a slot designed for the petri plate of the shearing device. Three equidistant points are marked from the centre of the cone using the motorized microscope stage. The vertical distance from each of these points to the micro-patterned substrate is determined using the microscope. The precision alignment screws on the fluid shear device are adjusted to eliminate possible tilt in the cone and obtain a constant vertical height for each of the marked points. This makes the axis of the cone perpendicular to the plate. The cone angle ß is less than 5°and the gap between the apex of the cone and petri plate is maintained less than the radius of the cone (Figure 5). When the cone alignment is done, the cone is raised and the dish with the substrate is placed beneath the cone plate and the cone is lowered down to a point where the cone apex starts deforming the cell. The substrate is transparent and hence the deforming of the cell can be monitored through the eye piece of the inverted microscope. The sample is grown on or adhered to the substrate. When the tip of the cone compresses the cell, the cone is raised by a few micrometers and the duty cycle of the pulse of the motor is increased to start the rotations in the cone. The tip of the cone is calibrated very close to the base plate at a distance ranging 10µm to 30µm between the tip and the substrate. The rotation of the cone is maintained at a range between 90 rpm and 800 rpm to generate a shear stress. The cone plate arrangement requires minimum amount of the fluid to apply fluid shear and hence a wastage of medium can be avoided.
Experimental
The device can be used for any adherent cell lines, primary cells, soft hydrogels, and tissues. In the present experiment 3T3 fibroblasts and human embryonic kidney (HEK) cells are used in the device for specific experiments to quantify the adhesion/ de-adhesion kinetics in cells and the role of the cytoskeleton in cells within constrained shapes.
The shear device is used to measure the adhesion strength of cells by quantifying the number of cells remaining on the substrate under different amounts of shear. A threshold value for the shear stress beyond which the detachment is initiated may be easily quantified using the master curve obtained by continually varying the shear stress in one experiment. The master curve, based on cellular detachment, gives a measure of the strength of interaction between the cells and the substrates. A pilot study using mouse fibroblasts (3T3) and human embryonic kidney cells (HEK) (Figure 9a) is conducted using the shear device. It is found that the detachment of HEK cells is faster than 3T3 cells based on comparing the half time and the decay constant between these cells. Such approaches are also used to modify the substrate with different extracellular matrix proteins to quantify the comparative properties of the substrate with different cells. Cancerous cells have different adhesive properties when compared to non-cancerous cells; a potential use of the device is to detect cancerous cell among the healthy cells. The adhesion de-adhesion kinetics via the master curve determines the mechanical differences between the healthy and cancerous cells.
The effect of cytoskeletal drugs like Blebbistatin, Taxol, Nocodazol on the adhesion strength of the cell is quantified using the shear device (Figure 9b). The drug Blebbistatin is an inhibitor for myosin II motor protein which is essential in the adhesion process. The adhesion de-adhesion studies are important in quantifying the effect of specific target molecules within the cell and its effect on the adhesion strength with the underlying substrate. Studies using 3T3 fibroblasts show an even distribution of cell spreading with detachment which follows a single exponential with time; this was absent for a control experiment on 3T3 cells which were not treated with the myosin II inhibitor, blebbistatin.
The shear device can also be used as a tool to study the shape modulation of a cell or the nucleus under shear flow. 3T3 cells transfected with vinculin GFP are cultured on micro-patterned coverslip coated with fibronectin. Focal adhesions in the cell are observed under static conditions (Figure 10a). However, when fluid shear stress of 1 pascal is generated in the cell environment for a duration of 30 minutes, change in cell shape and redistribution of the focal adhesion is noticed (Figure 10b). These assays are important for the study of changes in the mechanical properties of endothelium cells under flow.
The present invention thus provides a device which is light and compact and can be mounted on a standard inverted microscope for example confocal microscope, inverted fluorescence microscope, total internal reflection fluorescence microscope, polarization microscope, and the like. The device can generate shear stress on biological samples in a controlled environment The device is adapted for biological applications using live cells and tissues which require control of humidity, CO2 levels and temperature over a long term duration. The device combines the use of micro-patterned substrates to control cell shape and adhesion with the ability to apply shear stress. This allows for quantitative analysis of cell adhesion or any applications where adhesion to confined geometry is important. This also allows for de-adhesion measurements using the fluid shear device which can also be used as an assay to detect cancer like pathologies. The device can be fabricated at a significantly lower cost as compared to current commercially available rheometers and allows high resolution microscopy using existing standard microscopes.
The details of the invention has been exemplified using the fluid shear device on biological samples, however the device can also be used for generating shear stress on granular materials and other soft materials also.