Microfabricated topology platform and image analysis system for determining cell chirality

Abstract

Embodiments of methods, systems, and devices are disclosed for screening of cells for chirality. The methods include seeding and culturing cells onto a substrate having microgrooves, collecting images of the cells on the substrate, and performing an image analysis to determine the angular distribution of the cells with respect to the microgrooves. For some embodiments, the image analysis involves the use of fast Fourier transforms to determine the angular distribution. The methods, systems and devices provide improved means for rapidly evaluating cellular chirality.

Description

MICROFABRICATED TOPOLOGY PLATFORM AND IMAGE ANALYSIS SYSTEM FOR DETERMINING CELL CHIRALITYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 710,699, filed October 23, 2024, the disclosures of which are hereby incorporated by reference in their entireties.TECHNICAL FIELD

[0002] The present disclosure relates to screening systems and methods to test for environmental and genetic effects on cell chirality. Such screening systems and methods allow for the high throughput testing of drugs and genetic markers and how they may alter the chirality of different cell types.BACKGROUND

[0003] Chirality has recently been determined as an intrinsic property of cells that is heavily implicated in the development of left-right asymmetry organisms. While there is mounting interest in this field, there is still much to learn about the molecular mechanisms which govern this behavior and the tissue-wide consequences of failures in proper formation.

[0004] Cell chirality describes the intrinsic polarization of cells on the left-right (LR) axis and is considered a fundamental property of the cell, exhibiting biased morphology and motility across different morphological scales. The proper cell polarization involves normal cell viability and physiological functions. As a complex phenomenon consequent from the interplay of a variety of cell behaviors, cell chirality can act as a unique indicator or measure of the overall biological outcomes of cells when exposed to external stimuli, such as drugs and toxic environments. Potential applications of cell chirality characterization include drug screening and cytotoxicity determinations.

[0005] Current methods of assessing cell chirality are limited by complexity and are not well adapted to high throughput screening. Such methods include trapping cells between boundaries formed by microcontact printing or within matrigel bilayer systems.

[0006] Methods involving microcontact printing are limited because cell orientation occurs primarily at boundary walls, whereas the matrigel bilayer system is experimentally complex and involves sophisticated data analysis. Neither method is particularly well suited to high throughput screening analysis of environmental and chirality genetic determinants of cell chirality for different cell types. In order to advance progress, improved methods are required for high throughput screening of cell chirality.SUMMARY

[0007] Micro-topological cues or features on cell culture substrates have been widely used as quantitative characterization assays of multicellular chirality, such as ring- or stripshaped micropatterns, which provide geometrical confinements to the cell collectives and take the LR bias manifested in cell morphological alignments and migrational directions as readout for determining cell chirality. Although vigorous, these assays heavily rely on the micropatterned features that are commonly generated with extracellular matrix (ECM) proteins through microcontact printing technique, which could suffer from batch effect, time-consuming fabrication, as well as instability during storage, thereby limiting the throughput of cell chirality characterization while hindering the potential application of cell chirality as a biomarker in a readily standardized manner. To improve the robustness and efficiency of the current chirality assays for practical applications, this disclosure describes the design and fabrication method of a reusable high-throughput platform for cell chirality characterization, a power tool for studying the basic biological mechanism, and for drug screening applications.

[0008] Current systems and methods of assessing cell chirality include means and methods of trapping cells between boundaries formed by microcontact printing and within matrigel bilayer systems. In contrast, the microgroove system and method of this disclosure involves growing cells on substrates patterned with microgrooves, in such a manner that individual cells are not trapped, but rather can extend between neighboring microgrooves, sitting atop both the hills and the valleys of the microgrooves, thereby providing a system that is readily adapted to high throughput screening methods.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Fig. 1 A shows a top-down view of a patterned substrate according to the present disclosure.

[0010] Fig. IB shows a side view of the patterned substrate of Fig. 1A.

[0011] Fig. 2 shows a top-down view of a microtiter plate according to the present disclosure.

[0012] Fig. 3 shows a method of preparing a substrate of the present disclosure by photolithography.

[0013] Fig. 4 shows a system according to the present disclosure.

[0014] Fig. 5 shows the FFT analysis technique used to obtain the chiral coefficient

[0015] Fig. 6 shows HFF cells cultured on top of a substrate with microgrooves having 20 pm width and 5 pm depth.

[0016] Fig. 7 shows HFF cells cultured on top of a substrate with microgrooves having 50 pm width and 5 pm depth.

[0017] Fig. 8 shows HFF cells cultured on top of a substrate with microgrooves having 100 pm width and 5 pm depth.

[0018] Fig. 9 shows HFF cells cultured on top of a substrate with microgrooves having 200 pm width and 5 pm depth.

[0019] Fig.10 shows HFF cells cultured on top of a substrate with microgrooves having 50 pm width and 0.5 pm depth.

[0020] Fig. 11 shows HFF cells cultured on top of a substrate with microgrooves having 50 pm width and 1.0 pm depth.

[0021] Fig. 12 shows HFF cells cultured on top of a substrate with microgrooves having 50 pm width and 1.5 pm depth

[0022] Fig. 13 shows HFF cells cultured on top of a substrate with microgrooves having 50 pm width and 2.0 pm depth.

[0023] Fig. 14 shows chiral factors determined for various widths and depths.DETAILED DESCRIPTION

[0024] One embodiment of the present technology is a method of seeding and culturing cells onto a substrate, collecting images of the cells on the substrate, and analyzing the images to determine information related to cell chirality.

[0025] Referring now to Figs. 1A and IB are a top view and a side view, respectively, of the substrate 10. The substrate 10 has a top surface with an arrangement of microgrooves 12 having dimensions including a spacing 13 between neighboring microgrooves 12, a width 14 and a depth 16, the dimensions configured to allow individual cells 18 to bridge neighboring microgrooves 12.

[0026] High throughput methods for the analysis of cells on microgrooves involve handling many images or samples at once. The processing for these images needs to be accurate in determining the chiral bias of cells, simple for a user to implement, and computationally efficient. Previous approaches to determine cell chirality measure the gradient of phase contrast images to determine the local alignment of cells.

[0027] In order to facilitate high throughput screening, according to some embodiments of the present disclosure, a Fast Fourier Transform (FFT) is used to determine the degree of biased alignment from phase contrast images. A 2D FFT is applied to the image. A radial summation about the center of the image thus provides a plot of which angles occur the most within the image. Images of clear biased cell alignment display a curve whose peak is firmly on one side of the groove direction (0°). A positive (negative) angle is defined to represent counterclockwise (clockwise) alignment. The area under the curve is taken for the positive and negative sides. A chiral coefficient (CC) is determined by the following equation:_ Positive Sum-Negative SumPositive Sum+Negative Sum '

[0028] A positive (negative) CC value indicates that the global alignment of cells is generally CCW (CW). FFT results are validated by comparison to gradient analysis methods.

[0029] The analysis includes determining one or more parameters related to how the cells 18 are oriented with respect to the microgrooves 12. In particular, the analysis includes a determination of the angular distribution of cell orientation with respect to the microgrooves 12.According to some embodiments the images are analyzed by a gradient method providing local subregion alignment information. According to some embodiments, the one or more parameters includes a nematic order parameter. According to some embodiments, the analysis is performed by a fast Fourier transform method. According to some embodiments, the images are obtained by phase contrast microscopy.

[0030] Referring now to Fig. 2, according to some embodiments, the substrate 10 forms the bottom part of a microtiter plate 20. For such embodiments, the top surface of the substrate 10 is covered with a plurality of wells 22, each well of the plurality of wells being configured with water-tight boundaries 24 to hold liquid in place inside the wells 22 so that the liquid is in contact with a portion of the microgrooves 12. According to some such embodiments of the method, the cells in different wells of the plurality of wells have been subjected to different environmental conditions or have different genetic compositions. According to some embodiments, the different environmental conditions correspond to different concentrations of one or more drugs.

[0031] According to some embodiments, the microgrooves 12 have a width 14 between about 5 pm and about 200 pm and a depth 16 between about 0.2 pm and about 2 pm. According to some embodiments, the microgrooves have a width between about 20 pm and about 100 pm and a depth between about 0.5 pm and about 1.5 pm. According to some embodiments, the microgrooves have a width between about 5 pm and about 20 pm and a depth between about 0.2 pm and about 1.0 pm.

[0032] According to some embodiments, the substrate 10 is selected from the group consisting of silicate glass, cast polymers, plastic, hybrid silica glass, and glass composites.

[0033] According to some embodiments, the microgrooves 12 are fabricated through a method selected from the group consisting of photolithography with a patterned mask, laser engraving, electrochemical discharge machining, grinding-aided electrochemical discharge engraving, micromachining, abrasive waterjet machining, micromolding, nanoimprinting, injection molding, 3D printing, and combinations thereof.

[0034] According to some embodiments, a microtiter plate 20 is disclosed comprising a substrate 10 having a top surface with an arrangement of microgrooves 12 having a width 14 between about 5 pm and about 200 pm and a depth 16 between about 0.1 pm and about 2 pm, a plurality of wells 22 covering the top surface of the substrate 10, each well 22 having water-tightboundaries 24 to hold liquid in place so that the liquid is in contact with a portion of the microgrooves 12, wherein the wells 22 are configured to allow imaging of cells 18 attached to the top surface of the substrate 10 within the wells 22. According to some such embodiments, the substrate 10 of the microtiter plate 20 is selected from the group consisting of silicate glass, plastic, hybrid silica glass, and glass composites.

[0035] According to some embodiments the microgrooves 12 of the substrate 12 of the microtiter plate 20 is fabricated through a method selected from the group consisting of photolithography with a patterned mask, laser engraving, electrochemical discharge machining, grinding-aided electrochemical discharge engraving, micromachining, abrasive waterjet machining, micromolding, nanoimprinting, injection molding, 3D printing, and combinations thereof.

[0036] Referring now to Figs. 3A and 3B, methods are disclosed for fabrication of the substrate 10 by photolithography. According to the embodiment of Fig. 3A. the substrate 10 is formed directly by photolithography. As embodied in Fig. 3B, photolithography is used to create a mold for a cast polymer substrate 10. Both methods include steps A-D:Step A: Spin-coating photoresist 310 onto glass 315.Step B: Performing photolithography by covering the photoresist 315 with a mask and shining UV light on the exposed surface of the photoresist, thereby exposing glass surfaces having the desired spacing.Step C: Etching the exposed glass with an etchant such as hydrofluoric acid to the desired depth.Step D: Removing the photoresist with a photoresist remover solution, and rinsing to obtain a substrate (Fig. 3A) or the mold of a substrate (Fig. 3B).As embodied in Fig. 3A, the substrate 10 having microgrooves 12 with the desired width 14, depth 16, and spacing 13 is directly formed and the process continues:Step E: Seeding and culturing cells 18 onto the surface of the substrate 10, so that a portion of the cells 18 bridge neighboring microgrooves 12.As embodied in Fig. 3B, a mold of the substrate 10 is formed onto glass 315, and the process continues:Step E: Casting a polymer 320 onto the glass 315, which serves as a mold.Step F: Removing the cast polymer 320 from the glass mold to form the substrate 10 having microgrooves 12 with the desired width 14, depth 16, and spacing 13Step G: Seeding and culturing cells 18 onto the surface of the substrate 10, so that a portion of the cells 18 bridge neighboring microgrooves 12.

[0037] Referring now to Fig. 4, some embodiments provide a system 40 for screening cells 18 for chirality comprising the microtiter plate 20 described above, a pipetting means 42 for delivering liquids to the wells of the microtiter plate 20, a microscope 44 configured to image the top surface of the substrate 10 within the wells 22, and a processor 46 configured to analyze the image data to determine one or more parameters related to how the cells 18 are oriented with respect to the microgrooves 12, wherein the analysis includes a determination of an angular distribution of cell orientation with respect to the microgrooves 12.

[0038] For some embodiments of the system 40, the processor 46 is configured to analyze the image data by a gradient method providing local subregion alignment information. For some embodiments of the system 40, the processor 46 is configured to analyze the image data by a fast Fourier transform method. For some embodiments, the microscope 44 is configured to image the top surface of the substrate 10 by phase contrast microscopy.

[0039] For some embodiments of the system 40, the substrate is selected from the group consisting of silicate glass, plastic, hybrid silica glass, and glass composites.

[0040] For some embodiments of the system 40, the microgrooves 12 are fabricated through a method selected from the group consisting of photolithography with a patterned mask, laser engraving, electrochemical discharge machining, grinding-aided electrochemical discharge engraving, micromachining, abrasive waterjet machining, micromolding, nanoimprinting, injection molding, 3D printing, and combinations thereof.Examples:1. Microfabrication of grooved substrate

[0041] A cell culture substrate was prepared using microfabrication techniques. First, 76.2 mm diameter, 500 pm thick, double-side polished fused silica wafers (University Wafer cat# 1013) were cleaned and had the surface activated using piranha solution. The wafers were placed in a polytetrafluoroethylene (PTFE) holder and then in a freshly prepared bath of piranha solution consisting of a 1:1 ratio of 30% hydrogen peroxide and 96% sulfuric acid. The waferswere completely submerged for 15 minutes, then removed and immediately submerged in water. After 4 rinses in water, the wafers were dried with compressed air.

[0042] Each dried wafer was then patterned with the desired design using photolithography. To prepare the wafers for photolithography, each wafer was placed on a spin coater, and hexamethyldisilazane (HDMS) solution was added to cover about 75% of the center of the wafer. The wafer was then spun at 3000 rpm for 30 seconds to generate an even coating of HDMS. The spin coater was then paused for 30 seconds while SI 813 photoresist was added to cover about 75% of the wafer, then spun again at 3000 rpm for 30 seconds. The wafer was transferred to a 105C hot plate to warm the sample to 95C and bake for 1 minute before being transferred to a flat surface to cool to room temperature.

[0043] The wafers were then exposed to light using a broadband mid-UV light-emitting contact aligner (OAI). A mask with the desired pattern was loaded on the contact aligner, and the wafer was centered below the mask. The wafer was exposed to the UV light for an energy output of about 109 mJ / cm2. The wafer was then transferred to the S 1813 developer for 1 minute and then rinsed in circulating water for 1 minute before being dried with compressed air. The wafer was then hard-baked at 120C for 15 minutes. A Detak 8 contact profilometer was used to measure the step height between the surface of the photoresist and the exposed glass surface.

[0044] The patterned samples were then etched to the desired depth. First, the etch rate was confirmed using one wafer. The wafer was placed in a PTFE holder and then completely submerged in a buffered hydrofluoric acid solution for 5 minutes. The wafer was then immediately submerged in water, rinsed 4 times total, and dried with compressed air.

[0045] The step height between the top of the photoresist surface and the exposed glass was again measured with the Detak 8 profilometer. The original height measured before etching was then subtracted from this new height to obtain the etched depth. The etched depth was then divided by the etch time of 5 minutes to obtain the etch rate in microns per minute. This rate was then used to calculate the time to etch the wafers to the desired final groove depth.

[0046] The wafers were then etched to the desired groove depth. The wafers were placed in the PTFE holder and completely submerged in the buffered HF solution for the time calculated with the etch rate. The wafers were immediately submerged in water, rinsed 4 times, and dried with compressed air. The profilometer was used to measure the step height between thephotoresist and the exposed glass to confirm the height is equal to the original thickness before etching plus the desired groove depth.

[0047] The photoresist was then removed from the samples. The wafers were placed in a PTFE holder and submerged in a photoresist remover solution warmed to about 75C for 15 minutes. The wafers were removed from the solution, completely submerged in water for 4 total rinses, then dried with compressed air. The step height from the top of a groove to the bottom of a groove was measured with the profilometer to confirm the desired groove depth was achieved.

[0048] According to an alternative method, polymer substrate such as poly dimethyl siloxane (PDMS) or polymethylmethacrylate (PMA) can optionally be cast onto the etched substrate. The grooved polymer substrate can then be peeled off and fixed onto glass in preparation for assembly of the well system.2. Assembly of well system

[0049] The wells of a microtiter plate are formed on the glass wafer using 3D printing and PDMS. Specifically, a 3D model of the well system mold is generated using SolidWorks, and then 3D printed using a SLA 3D printer (Form 2, Formlabs) with clear resin (RS-F2-GPCL-04, Formlabs). PDMS (Sylgard 184, Dow-Corning) is prepared by mixing the base and curing agent in a 10:1 ratio, and subsequently poured into the well system mold. After degassing under vacuum, the mold is transferred to a 60°C oven and cured for 6 hours. The cured PDMS wells are then peeled from the mold, trimmed, and prepared for adhesion to the glass wafer. A diluted version of PDMS is used as an adhesive to bond the well system to the glass wafer. Briefly, PDMS is mixed as previously described and then diluted with hexane in a 2:3 ratio. The diluted PDMS is poured into a petri dish lined with aluminum foil. The bottom of the well system is dipped into the PDMS, and it is adhered to the glass wafer after the hexane evaporates. Similarly, the PDMS adhesive involves 6 hours of baking in an oven before the well system is ready for subsequent use.

[0050] An alternative method involves using a commercially available bottomless sticky slide (ibidi). According to the product information, the adhesive base can be directly affixed to the glass wafer.3. Microgroove device cleaning and sterilization

[0051] The assembled microgroove device can be used for further cleaning and sterilization. To clean the device, we treated it with 10 ml of bleach (10%) solution for 10 minutes, followed by rinsing the wells with DI water. Add soap water solution to completely remove the bleach from the device, after completely cleaning the surface, rinse with DI water. Furthermore, we started with a series of organic solvent treatments to remove the debris and organic and inorganic substituents from the device. To perform this, we added 70% ethanol solution (5-7 mL), waited another 5 minutes, and sonicated for 5 minutes in 30 amplitudes, and rinsed with DI water. Next, the device is treated with acetone solution (5-7 mL) for 5 minutes, sonicated for 5 minutes at 30 amplitudes, and rinsed with DI water. Next, the device is filled with isopropanol (5-7 mL) in the wells, waited for 5 minutes, and sonicated for 5 minutes at 30 amplitudes. Finally, the well system is filled with 70% ethanol (5-7 mL) to the wafers and waited for 5 minutes. Carefully remove the device and blow N2 air into the surface of the wells to completely dry the ethanol. To perform the sterilization, we kept the device in UV ozone exposure (be sure to check the grooved surface to expose it to UV lights) for 15-30 minutes. Deozone the UV ozone chamber for 10 minutes and remove the device for UV ozone exposure.4. Cell culture and imaging

[0052] On the day of cell culture, the wells are filled with Fibronectin (50 pg / mL) solution for 30 minutes to coat the microgrooves. The Fibronectin solution is carefully removed from the well system, the wells are filled with the growth media, and the system is incubated for 30 minutes.

[0053] Depending on the cells (HFF, C2C12, HUVEC, and AC 16) to be cultured, the cell cultures are prepared to contain lxl0?cells / wells and added uniformly to the wells (100-150 pL), the device is swirled to distribute the seeded cells to spread the entire surface and incubated at 37 °C for 15 minutes. After 30 minutes, cell adhesion patterns are checked on the well surface. It was found that the seeded cells are attached 80-90%. The media was discarded using a pipette (from the edges of the wells, without touching the well surface) and 150 pl of fresh growth media (DMEM with 10% FBS) 150 was added. The incubation was continued for 24 or 48 h, depending on cell type.

[0054] To perform the fluorescence, staining, the cells on the microgrooves were fixed with 4% paraformaldehyde solution for 20-30 minutes. Next, the fixed cells were blocked andpermeabilized with 0.1% triton XI 00, containing 3% bovine serum albumin (BSA) in PBS for 1 h. Then, the blocked cells were stained with Phalloidin (actin antibody, 1 :300 dilution) and incubated for 1 h. Next, the stained cells were washed using PBS containing 0.02% Tween 20 solution 3 times, 15 mins each. Next, the cell nuclei were stained with DAPI (1:1000 dilution), for 30 minutes, and washed with PBS containing 0.02% Tween 20 solution 3 times, 15 mins each. To image the cells by fluorescence imaging, depending on the fluorescent-tagged (Alexa 488, 594, 647, etc) antibody, we employed the specified filters (green or red) in the microscope. The images were acquired at different wells and random locations in each well using 10X magnification. The phase contrast and fluorescence, and its overlay ed images (minimum of 20-25 images per group) were acquired at multiple places. The acquired images were analyzed using MATLAB code to observe the chiral bias and cell alignments on the microgrooves.5. Determining if microgrooves are suitable for assessing cell chirality.Referring now to Fig. 5, HFF cells were seeded onto glass micro-grooved substrates, and phase contrast images were collected 510. A 2D FFT provides a power spectrum 520. Radial summation provides an angular distribution of cells 530. Obtaining the positive and negative areas under the curve then allows the chiral coefficient to be calculated 540 according to equation (1). The direction of the groove is set to be 0°. A positive angle of alignment is considered counterclockwise (CCW), while a negative angle is considered clockwise (CW). These assignments are determined by convention. Groove images were first analyzed with the gradient approach, and cells were determined to have a counterclockwise (CCW) bias. The FFT approach yielded the same determinations with a trend that is almost identical to the gradient-based approach. This indicates that the platform is effective in viewing chirality and that the analysis approach is effective. There are several advantages and disadvantages to note with both approaches. The gradient approach utilizes local subregion information to generate a global assessment of alignment. Obtaining many local vectors allows for the generation of other metrics, such as the nematic order parameter. This can be time consuming and can add up if analyzing hundreds of images. The estimated analysis time for each image is 10-20 seconds (1920x1440 resolution). The FFT approach is much quicker, taking only 3-5 seconds per image. The net result is additionally the same in terms of the trend that is developed. However, the metric obtained is less statistically robust than the gradient approach. Hundreds of alignment vectors are obtained togenerate a mean angle that can be tested for significance. The FFT approach only generates 1 chiral factor per image that describes the global alignment.

[0055] Referring now to Figs. 6-9, human foreskin fibroblast (HFF) cells are shown seeded and grown on substrates with microgrooves 12 having a depth 16 of 2 pm and widths 14 increasing from 50 pm for Fig. 6 to 100 pm for Fig. 7, to 150 pm for Fig. 8, to 200 pm for Fig. 9. For all of Figs. 6-9, the white scale bar is 100 pm in length. As can be discerned from these Figs., for a depth 16 of 2 pm, the HFF cells are confined within the microgrooves 12 and are unable to span between neighboring microgrooves 12. Under these circumstances, the HFF cells tend to orient along the walls of the microgrooves 12. As discussed further below, at widths 14 below about 50 pm, cells are limited in rotational motion, whereas for widths 14 above about 100 pm, the cells have room to orient along the walls, but are less oriented towards the center of the microgrooves 12.

[0056] Referring now to Figs. 10-13, the effect of varying the depth 16 of the microgrooves 12 for a constant width 14 of 50 pm. For all of Figs. 10-13, the white scale bar is 50 pm in length. For Figs. 10-13, the depths increase from 0.5 pm to 1.0 pm, to 1.5 pm, to 2.0 pm, respectively. As can be discerned from Figs. 10 and 11, for depths 16 of 0.5 pm and 1.0 pm, the HFF cells are able to orient and to span across microgrooves 12, whereas the cells are partially or fully confined within the microgrooves 12 having depths 16 of 1.5 pm and 2.0 pm.

[0057] Fig. 14 summarizes measurements of chiral factors obtained by FFT analysis as a function of microgroove width 14 and depth 16 for the HFF cells. The positive numbers indicate generally counterclockwise alignment, and the larger numbers indicate a greater overall degree of orientation. Notably, for HFF cells, modest depths of 0.5 pm to 1.5 pm and widths in the range of 20 pm to 100 pm correlate with the highest degree of orientation. For the HFF cells, depths of 0.1 pm are too shallow to provide good orientation, whereas for depths of 2 pm or more cells are trapped within microgrooves, likewise inhibiting good orientation.

[0058] As used herein, the term “about” means within 10%.

[0059] Although the invention has been described and illustrated with respect to exemplary embodiments thereof, it should be understood by those skilled in the art that the foregoing and various other changes, omissions and additions may be made therein and thereto, without parting from the spirit and scope of the present invention.

Claims

What is claimed is:

1. A method of screening cells for chirality comprising:seeding and culturing cells onto a substrate, the substrate having a top surface with an arrangement of microgrooves configured to allow individual cells to bridge neighboring microgrooves;collecting images of the cells on the substrate;performing an analysis of the images to determine one or more parameters related to how the cells are oriented with respect to the microgrooves, wherein the analysis includes a determination of an angular distribution of cell orientation with respect to the microgrooves.

2. The method according to claim 1, wherein the microgrooves have a width between about 5 pm and about 200 pm and a depth between about 0.2 pm and about 2.0 pm.

3. The method according to claim 1, wherein the microgrooves have a width between about 20 pm and about 100 pm and a depth between about 0.5 pm and about 1.5 pm.

4. The method according to claim 1, wherein the microgrooves have a width between about 5 pm and about 20 pm and a depth between about 0.2 pm and about 1.0 pm.

5. The method according to any one of claims 1-4, wherein the analysis is performed by a gradient method providing local subregion alignment information.

6. The method according to claim 5, wherein the one or more parameters includes a nematic order parameter.

7. The method according to any one of claims 1-6, wherein the substrate is selected from the group consisting of silicate glass, cast polymer, plastic, hybrid silica glass, and glass composites.

8. The method according to any one of claims 1-4 or 7, wherein the analysis is performed by a fast Fourier transform method.

9. The method according to any one of claims 1-8, wherein the images are obtained by phase contrast microscopy.

10. The method according to any one of claims 1-9, wherein the microgrooves are fabricated through a method selected from the group consisting of photolithography with a patterned mask, laser engraving, electrochemical discharge machining, grinding-aided electrochemical discharge engraving, micromachining, abrasive waterjet machining, micromolding of a polymer substrate, nanoimprinting, injection molding, 3D printing, and combinations thereof.

11. The method of any one of claims 1-10, wherein the top surface of the substrate is covered with a plurality of wells, each well of the plurality of wells being configured with water-tight boundaries to hold liquid in place so that the liquid is in contact with a portion of the microgrooves.

12. The method of claim 11, wherein the cells in different wells of the plurality of wells have been subjected to different environmental conditions or have different genetic compositions.

13. The method of claim 12, wherein the different environmental conditions correspond to different concentrations of one or more drugs.

14. A microtiter plate comprising:a substrate having a top surface with an arrangement of microgrooves having a width between about 5 pm and about 200 pm and a depth between about 0.1 pm and about 2 pm; a plurality of wells covering the top surface of the substrate, each well having water-tight boundaries to hold liquid in place so that the liquid is in contact with a portion of the microgrooves, wherein the wells are configured to allow imaging of cells attached to the top surface of the substrate within the wells.

15. The microtiter plate of claim 14, wherein the substrate is selected from the group consisting of silicate glass, plastic, hybrid silica glass, and glass composites.

16. The microtiter plate of either claim 14 or claim 15, wherein the microgrooves are fabricated through a method selected from the group consisting of photolithography with a patterned mask, laser engraving, electrochemical discharge machining, grinding-aided electrochemical dischargeengraving, micromachining, abrasive waterjet machining, micromolding, nanoimprinting, injection molding, 3D printing, and combinations thereof.

17. A system for screening cells for chirality comprising:the microtiter plate of any one of claims 14-16;a pipetting system for delivering liquids to the wells of the plate;a microscope configured to image the top surface of the substrate within the wells; and a processor configured to analyze the image data to determine one or more parameters related to how the cells are oriented with respect to the microgrooves, wherein the analysis includes a determination of an angular distribution of cell orientation with respect to the microgrooves.

18. The system of claim 17, wherein the processor is configured to analyze the image data by a gradient method providing local subregion alignment information.

19. The system of claim 17, wherein the processor is configured to analyze the image data by a fast Fourier transform method.

20. The system of any one of claims 17-19, wherein the microscope is configured to image the top surface of the substrate by phase contrast microscopy.

21. The system of any one of claims 17-20, wherein the substrate is selected from the group consisting of silicate glass, plastic, hybrid silica glass, and glass composites.

22. The system of any one of claims 17-21, wherein the microgrooves are fabricated through a method selected from the group consisting of photolithography with a patterned mask, laser engraving, electrochemical discharge machining, grinding-aided electrochemical discharge engraving, micromachining, abrasive waterjet machining, micromolding, nanoimprinting, injection molding, 3D printing, and combinations thereof.

Images