Automated cell culture scratching apparatus and method

Abstract

The present disclosure provides a device comprising a frame, a 3-axis gantry operably coupled to the frame, a scratch tip tool removably coupled to the 3-axis gantry, a sample holder operably coupled to the frame configured to receive a culture vessel containing cells, and at least one processing unit. The processing unit is configured to receive information comprising a target pattern and cause the scratch tip tool to scratch the cells in the target pattern. The device enables automated and precise creation of scratch patterns in cell cultures for wound healing and migration assays.

Description

[0001]Princeton - 101476 AUTOMATED CELL CULTURE SCRATCHING APPARATUS AND METHOD CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 63 / 656,861, titled Scalable Cell Resection Apparatus To Precisely Cut and Pattern Living Tissues, filed June 6, 2024, which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No.: GM133574 awarded by the National Institute of Health. FIELD OF INVENTION The present disclosure relates to automated cell culture manipulation systems, and more particularly to a programmable robotic apparatus for precisely scratching and patterning living tissue cultures. BACKGROUND Cell migration and wound healing are fundamental biological processes that play crucial roles in various physiological and pathological conditions. Understanding these processes is essential for advancing medical research, developing therapeutic interventions, and improving patient outcomes. In vitro assays have become valuable tools for studying cell migration and wound healing in controlled laboratory settings. One widely used method for investigating these processes is the scratch assay, also known as the wound healing assay. This technique involves creating a gap or "wound" in a confluent cell monolayer and observing how cells migrate to close the gap over time. The scratch assay provides insights into cell behavior, migration rates, and factors that influence wound closure. Traditional scratch assays typically involve manually creating a scratch or gap in the cell monolayer using a pipette tip or similar tool. While this approach has been widely adopted, it presents several challenges. Manual scratching can lead to variability in scratch width and shape, making it difficult to standardize experiments and compare results across different trials or laboratories. Additionally, the manual nature of the technique limits throughput and can be time-consuming when performing multiple experiments. Princeton - 101476 Efforts to improve upon the traditional scratch assay have led to the development of various alternative methods and tools. These include the use of physical barriers, such as silicone inserts, to create cell-free zones, as well as more advanced techniques like laser ablation or microfluidic devices. While these approaches offer certain advantages, they often require specialized equipment or materials that may not be readily available in all research settings. There remains a need for improved methods and systems that can overcome the limitations of manual scratch assays while maintaining accessibility and ease of use. Ideally, such solutions would provide increased reproducibility, higher throughput, and greater flexibility in creating diverse wound patterns. Additionally, the ability to precisely control and automate the scratching process could open up new possibilities for studying complex cellular behaviors and interactions. SUMMARY This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. According to an aspect of the present disclosure, a device is provided. The device includes a frame. The device includes a 3-axis gantry operably coupled to the frame. The device includes a scratch tip tool removably coupled to the 3-axis gantry. The device includes a sample holder operably coupled to the frame configured to receive a culture vessel containing cells. The device includes at least one processing unit configured to receive information comprising a target pattern and cause the scratch tip tool to scratch the cells in the target pattern. According to other aspects of the present disclosure, the device may include one or more of the following features. The 3-axis gantry may be configured to have belt-driven XY axes and a servo or solenoid-driven Z-axis. The culture vessel may be a Petri-style dish or a multiwell plate. The scratch tip tool may comprise a 20 μL or smaller pipette. The scratch tip tool may be removably coupled to a scratch tip tool holder. The scratch tip tool holder may be removably coupled to the 3-axis gantry. The device may further comprise an alignment ring removably coupled to the laboratory dish or plate. The at least one processing unit may be further configured to control one or more motors of the 3-axis gantry to position the pipette tip tool and adjust a scratching speed of the pipette tip tool based on properties of the cells. The at least one processing unit may be further configured to control the motors to perform multiple passes of the pipette tip tool over a scratch area to ensure complete cell clearance. The at least one Princeton - 101476 processing unit may be further configured to generate a raster pattern for the scratch tip tool to create one or more geometric shapes in the cells. According to another aspect of the present disclosure, a method for scratching a tissue is provided. The method includes providing a device comprising a frame, a 3-axis gantry operably coupled to the frame, a scratch tip tool removably coupled to the 3-axis gantry, and a sample holder operably coupled to the frame. The method includes receiving, by at least one processing unit, information comprising a first target pattern. The method includes causing, by the at least one processing unit, the scratch tip tool to scratch first cells in the first target pattern to form a first scratch pattern in the first cells, where the first cells are disposed in a culture vessel that is in contact with the sample holder. According to other aspects of the present disclosure, the method may include one or more of the following features. The method may further comprise establishing an origin by aligning a tip of the pipette tip tool with a target location on an alignment ring on the culture vessel. The method may further comprise attaching the alignment ring to the culture vessel prior to establishing the origin. The method may further comprise washing the culture vessel with a buffered solution after scratching the first cells, adding a suspension comprising second cells to the culture vessel over the first cells, and forming a co-culture by incubating the culture vessel containing the first cells and the second cells. The first cultured cells and the second cultured cells may comprise different cell types. The method may further comprise scratching a second pattern in the co-culture and adding third cells to the second scratched pattern. The first target pattern may comprise a spiral, grid, or a geometric shape. The method may further comprise adjusting culture conditions to modulate cell-cell adhesion between the first cells and second cells. The method may further comprise imaging the co-culture to analyze spatial organization and interactions between different cells. The first cells may comprise keratinocytes and the second cells may comprise fibroblasts. The method may further comprise staining the first cells prior to scratching and staining the second cells prior to adding the suspension to the culture vessel. The method may further comprise fixing the culture vessel containing the first cells and the second cells after incubation. Causing the scratch tip tool to scratch the first cells may comprise controlling one or more motors of the 3-axis gantry to position the scratch tip tool and adjusting a scratching speed of the scratch tip tool based on properties of the first cells. Causing the scratch tip tool to scratch the first cells may further comprise controlling the motors to perform multiple passes of the pipette tip tool over a scratch area to ensure complete cell clearance. Causing the scratch tip tool to scratch the first cells may Princeton - 101476 further comprise generating a raster pattern for the scratch tip tool to create one or more shapes in the first cells. The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive. BRIEF DESCRIPTION OF FIGURES Non-limiting and non-exhaustive examples are described with reference to the following figures. FIG. 1 illustrates an isometric view of a device for performing automated scratch assays on cultured cells, according to aspects of the present disclosure. FIG.2A-2C depict schematic representations of a scratch assay performed by a scratch tip (2A) moving across a cell monolayer (2A), leaving a cell-depleted region (wound, see 2B), following by a healing process (2C). FIG.3A and 3B show phase-contrast images of a scratch pattern in a Petri dish, where the scale bar is 2 mm, Figure 3B showing a zoomed in image at the location shown by the box in Figure 3A. FIG. 4 is an image showing 12 linear scratches made by a machine according to an embodiment, where the scale bar is 2 mm. FIG. 5A and 5B relate to scratch uniformity on a keratinocyte monolayer, showing captured images of the scratches (5A) and a graph showing width error measurements across the two scratch types (5B). The scale bars in 5A are 1 mm. FIG. 6 shows a graph of wound healing rate over time following a scratch, according to an embodiment. FIG.7 is a graph showing width error measurements across different scratch types for scratches generated on a Madin-Darby canine kidney (MDCK) monolayer without calcium chelation, including by hand, by machine at a low speed, and by machine at a high speed. FIG. 8 is an image showing a demonstration of area clearance from tip overlap, where the programmed path diameters are 3, 2, 1, and 0.5 mm, and the scale bar is 2 mm. FIG.9A and 9B shows a schematic of a raster mechanism (9A) and calculation of raster overlap (9B). FIG. 10A and 10B are images of square-shaped regions generated either via positive area clearance (10A) or negative area clearance (10B), where the scale bar is 2 mm. Princeton - 101476 FIG.11A and 11B are images of a complex target shape (11A) achieved through restoring, and the resulting scratch pattern in the cells (11B), where the scale bar is 2 mm. FIG.12 shows a schematic for co-culturing via scratching and backfilling. FIG. 13A and 13B are fluorescence images of a spiral pattern scratched into first cells, then backfilled with second cells, where the first and second cells are stained different colors; the scale bar in 13A is 2 mm, while the scale bar in 13B is 500 mm. FIG.14 is an image showing scratch resolution using a 10 µL pipette tip. Scale bar is 5 mm. DETAILED DESCRIPTION The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein. Advancements in robotics and automation technologies have the potential to address many of the challenges associated with traditional scratch assays. By leveraging precise mechanical control and programmable systems, it may be possible to create more consistent and reproducible wounds in cell cultures. Such approaches could not only improve the reliability of experimental results but also enable the exploration of more complex wound geometries and patterns that are difficult to achieve manually. Furthermore, the integration of automation with cell culture techniques could extend beyond simple wound creation. There is potential for developing systems that can perform multiple steps in the experimental workflow, such as creating wounds, capturing images, and even analyzing results. This level of automation could greatly increase the efficiency and throughput of cell migration and wound healing studies. As research in cell biology and regenerative medicine continues to advance, there is an ongoing need for tools and techniques that can provide more detailed and nuanced insights into cellular behavior. Improved methods for studying cell migration and wound healing in vitro have the potential to accelerate discoveries in fields such as cancer research, tissue engineering, and drug development. The "scratch assay" (see FIGS. 2A-2C) - dragging a pipette tip (see FIG. 2A) or sharp object through a cultured tissue to create a “wound” (a cell-depleted region) (see FIG. 2B) and monitoring the cellular healing response (see FIG.2C) in the resulting gap - is among the most common approaches to study cell migration and healing in vitro but also, perhaps, among the least reproducible and scalable due to the manual nature of the process. Princeton - 101476 While a popular protocol paper on the manual method has nearly 5,000 citations at this point, and the method is largely free, the traditional scratch assay relies on pressure, tool orientation and brand, speed, and manual stability and is inherently limited in precision, throughput, and scalability (e.g., it is more difficult in a 96-well plate than in a 6-well plate). Moreover, there is a missed opportunity to use "scratching" as a form of subtractive manufacturing to produce much more complex tissue geometries and easily prepare unique systems-level co-cultures. Given the ubiquity and importance of scratch assays, new approaches improving the reproducibility, throughput, and versatility can benefit a broad range of research fields. While alternative solutions to generate gaps in tissues are well represented in the literature, none of them address all of the challenges. One popular approach is the "barrier removal assay," where cells are seeded on either side of a rubber stencil, and then the stencil is removed to generate a "gap." While versatile, the approach requires precision pipetting and simply does not scale to small culture vessels. Commercial rubber inserts are available but are limited in geometry and configuration and are costly consumables. Further, there is a concern that barrier removal may not properly damage the surrounding tissue consistent with actual injury. Similarly, DIY (do-it-yourself) parallel scratchers based on machined or molded tips have been used effectively in multi-well plate studies, but the approach relies on sophisticated machine- shop CNC (computer numerical control) capabilities, still requires user-applied pressure and speed, can only make straight lines, and is intrinsically limited to a single specific substrate (e.g., 96-well plates only). While commercial scratch systems exist, they are also limited to only a few well plate options (e.g., 24 / 96 well) and straight lines, and the cost is prohibitively high, relatively speaking (~$10,000 - $20,000 at the time of writing). A variety of alternative approaches to remove cells and tissue also exist that are based on the use of microfluidics. These approaches typically rely either on spatially dosing enzymes such as trypsin for controlling cell removal or physically removing cells based on pneumatic membrane compression removal. While precise and reproducible, these methods require expensive microfluidics manufacturing capabilities, are not programmable without creating new devices, and are relatively inaccessible to the broader community that performs scratch assays due to the required expertise and infrastructure. Hence, there is an exciting opportunity to redevelop the common, mechanical form of the scratch assay around flexible, programmable, open- source hardware that can be adopted by any laboratory. The present disclosure relates to an automated cell culture scratching apparatus and method. This apparatus and method may provide improved precision, reproducibility, and efficiency for performing scratch assays and related cell culture experiments. Princeton - 101476 In some cases, the automated scratching apparatus may be compact and portable, allowing for use directly within a sterile culture hood environment. This capability may help maintain sterile conditions throughout the experimental process. The automated nature of the apparatus may enable more consistent and repeatable scratching patterns compared to manual techniques. By reducing variability between experiments, the apparatus may improve the reliability and comparability of results in cell migration and wound healing studies. In some implementations, the apparatus may be designed with cost-effectiveness in mind. The components and materials used in construction may be selected to keep production costs low while maintaining functionality. In some cases, the total cost to produce the apparatus may be less than $500. The method enabled by this apparatus may allow for precise control over scratch parameters such as depth, width, and pattern geometry. This level of control may open up new possibilities for studying complex wound healing dynamics and cell migration behaviors. By automating the scratching process, the apparatus and method may also increase experimental throughput. Multiple samples or replicates may be processed more quickly and consistently than with manual techniques. In some cases, the apparatus may be programmable, allowing researchers to define custom scratch patterns tailored to specific experimental needs. This flexibility may enable novel experimental designs that were previously impractical or impossible with manual methods. The automated scratching apparatus and method may have applications across various fields of cell biology research, including but not limited to wound healing, cancer metastasis, and tissue engineering. By providing a more standardized and versatile approach to scratch assays, this technology may accelerate progress in these important areas of study. FIG. 1 illustrates an isometric view of a device 100 for performing automated scratch assays on cultured cells. The device 100 includes a frame 102 that provides structural support for the components. A 3-axis gantry system 104 is operably coupled to the frame 102 and enables controlled movement in the x, y, and z directions. In some cases, the 3-axis gantry system 104 may be configured to have belt-driven XY axes and a servo or solenoid-driven Z-axis. A scratch tip tool 106 is removably coupled to the 3-axis gantry system 104 for creating scratch patterns in cell cultures. In some cases, the scratch tip tool 106 may include any appropriate component for creating a depletion zone when moved through a group of cells. Non-limiting examples include, e.g., a pipette tips (including, e.g., a 0.1 µL – 10 µL, a 0.1 µL - 20 µL pipette tip, a 20 µL - 200 µL pipette tip, or 200 µL-1000 µL pipette tip; preferably a 20 µL or smaller Princeton - 101476 pipette tip), needles (e.g., 10 gauge needles or smaller, such as 10-34 gauge needles), wires (e.g., 1-30 gauge wires), etc. The outer diameter of the scratch tip needs to be of an appropriate size for the target pattern desired within the cells, but can otherwise be any appropriate size. The scratch tip tool 106 may be removably coupled to the 3-axis gantry system 104 directly. The scratch tip tool 106 may be removably coupled to a scratch tip tool holder 118. The scratch tip tool holder 118 may be removably coupled to the 3-axis gantry system 104. In some implementations, the scratch tip tool holder 118 may use an adhesive (such as an adhesive putty) to secure the scratch tip tool. In some implementations, the scratch tip tool holder 118 may use a mechanical lock or clamp to secure the scratch tip tool. The device 100 includes a sample holder 108 that is operably coupled to the frame 102 and configured to receive a culture vessel 110 containing cultured cells. The sample holder 108 may be a customized plate holder that can accommodate different types of culture vessels from 3.5 cm dishes to 96-well plates. In some cases, the culture vessel 110 may be a Petri-style dish or a container having multiple locations for containing cells (e.g., a multi-well array). A processor 112 controls the operation of the device 100. The processor may include one or more processing units that may work to collectively control operation of the device. Some or all of the processing units may be located on the device 100. Some or all of the processing units may be located remotely (e.g., may be coupled to the device via one or more wires, may communicate wirelessly, etc.). In some implementations, an communications interface 120 may be provided that is operably coupled to an on-board processor. The communications interface may include, e.g., a physical port such as an ethernet port, a USB port, etc., and / or may include a wireless interface for one or more wireless protocols. The processor 112 may be configured to receive information comprising a target pattern and cause the scratch tip tool 106 to scratch the cells in the target pattern. The processor 112 coordinates with motors 114 and 116 to precisely position the scratch tip tool 106 relative to the culture vessel 110. In some implementations, the device 100 may further comprise an alignment ring removably coupled to the culture vessel 110. This alignment ring may assist in establishing a reference point for the scratch patterns. The device 100 may be controlled using free drawing software, such as Inkscape, which allows for programming of custom scratch patterns. This software interface enables researchers to define and execute complex scratch geometries tailored to specific experimental needs. In some cases, the method may involve establishing an origin by aligning a tip of the scratch tip tool with a target location on an alignment ring on the culture vessel. The alignment ring Princeton - 101476 may be attached to the culture vessel prior to establishing the origin. This alignment process may help ensure consistent and accurate positioning of the scratch patterns across multiple experiments. The method may include receiving information comprising a first target pattern by at least one processing unit. The first target pattern may comprise a spiral, grid, or a geometric shape. The at least one processing unit may be configured to control one or more motors of the 3-axis gantry to position the scratch tip tool based on the received pattern information. In some implementations, the at least one processing unit may be configured to adjust a scratching speed of the scratch tip tool based on properties of the cells. For example, the scratching speed may be increased for cells with stronger adhesion properties to ensure complete removal. The method may involve causing the scratch tip tool to scratch first cells in the first target pattern to form a first scratch pattern in the first cells. In some cases, the at least one processing unit may be configured to control the motors to perform multiple passes of the scratch tip tool over a scratch area to ensure complete cell clearance. Additionally, the at least one processing unit may be configured to generate a raster pattern for the scratch tip tool to create one or more geometric shapes in the cells. After scratching, the culture vessel may be washed with a buffered solution to remove cell debris. In some implementations, the first cells may be stained prior to scratching to aid in visualization and analysis of the scratch pattern. The method may further include adding a suspension comprising second cells to the culture vessel over the first cells. In some cases, the second cells may be stained prior to adding the suspension to the culture vessel. This approach may allow for differentiation between the first and second cell populations. A co-culture may be formed by incubating the culture vessel containing the first cells and the second cells. The incubation conditions may be adjusted to modulate cell-cell adhesion between the first cells and second cells. In some implementations, the method may involve scratching a second pattern in the co- culture. This second scratching step may allow for the creation of more complex multi-cell type arrangements. After creating the second pattern, third cells may be added to the second scratched pattern, further increasing the complexity of the cell culture system. The method may include imaging the co-culture to analyze spatial organization and interactions between different cells. This imaging step may provide valuable insights into cell behavior and interactions in the patterned co-culture system. Princeton - 101476 In some cases, the culture vessel containing the first cells and the second cells may be fixed after incubation. Fixation may help preserve the spatial arrangement and cellular structures for further analysis or long-term storage. The method steps described may be applied to various cell types and experimental designs. For example, in some implementations, the first cells may comprise keratinocytes and the second cells may comprise fibroblasts, allowing for the study of skin-related cellular interactions and wound healing processes. In some cases, the automated scratching apparatus may employ a raster cutting technique to gradually remove tissue and produce complex patterns. This technique may involve moving the scratch tip tool back and forth in overlapping passes to clear a defined area of cells. The raster pattern may be generated by controlling the motors to move the scratch tip tool in a series of parallel lines with a specified overlap between each pass. The overlap percentage may range from about 50% to about 90% of the scratch tip tool width to ensure complete clearance of cells. The raster cutting technique may allow for the creation of various geometric shapes and patterns in cell cultures. For example, circular patterns may be created by gradually increasing or decreasing the radius of the raster pattern. In some implementations, the raster pattern may be used to create positive or negative space designs, where either the cleared area or the remaining cell area forms the desired pattern. The automated scratching apparatus may be utilized to create co-cultures by scratching one cell type and backfilling with another cell type. This process may involve first creating a scratch pattern in a monolayer of a first cell type, then adding a suspension of a second cell type to fill the cleared areas. The types of cells that may be used in this co-culture method may include, but are not limited to, keratinocytes, fibroblasts, endothelial cells, smooth muscle cells, and various types of stem cells. In some cases, the first cell type may be stained prior to scratching to aid in visualization and differentiation from the second cell type. Non-limiting examples of stains that may be used include fluorescent membrane dyes, including lipophilic carbocyanine dyes such as DiI, DiO, or DiD, as well as cytoplasmic stains like CellTracker dyes. The second cell type may also be stained with a contrasting dye before being added to the culture. The scratching speed of the automated apparatus may be adjustable, with the ability to operate at various speeds based on the capabilities of the motors, actuators, etc., that control the device. In some implementations, the velocity of the scratch tip (vtip) may be, e.g., 0 < vtip≤ 500 mm / s. In some implementations, the velocity may be required to remain constant while scratching Princeton - 101476 a pattern. In some implementations, the velocity may be configured to vary during operation (for example, a relatively slow speed in a first well and a relatively fast speed in a second well), vary while scratching a pattern through a single group of cells (in some implementations, multiple passes at different speeds may be used to achieve optimal cell clearance; for example, an initial high-speed pass may be followed by a slower, more precise pass to ensure complete removal of cells in the target area), or even vary during a single directional scratch (e.g., a first speed at the start of a linear scratch and a second speed at the end of the same linear scratch). The optimal scratching speed may vary depending on the properties of the cell type being scratched, such as adhesion strength and monolayer integrity. For cells with stronger adhesion, higher speeds may be used to ensure complete removal, while lower speeds may be more appropriate for more delicate cell types. The method may include a fixation step after the co-culture has been established. Any appropriate fixing agents may be used. Fixing agents that may be used include, but are not limited to, paraformaldehyde (typically at concentrations of 2-4%), glutaraldehyde (0.5-2%), or methanol. The choice of fixing agent may depend on the specific cell types used and the intended downstream analysis methods. In some cases, the co-culture conditions may be adjusted to modulate cell-cell adhesion between the first and second cell types. This may involve altering the calcium concentration in the culture medium, with concentrations ranging from about 0.05 mM to about 2 mM depending on the desired level of adhesion. Other factors that may be adjusted include the presence of adhesion-promoting or adhesion-inhibiting molecules in the culture medium. The automated scratching apparatus may also be used to create more complex multi-layered cultures by performing additional scratching and cell addition steps. For example, after creating an initial co-culture, a second scratch pattern may be created, followed by the addition of a third cell type. This process may be repeated to create intricate arrangements of multiple cell types within a single culture. In some cases, the automated scratching apparatus and method may be used to create complex co-cultures with different cell types. For example, the first cells may comprise keratinocytes and the second cells may comprise fibroblasts. This combination may allow for the study of interactions between epidermal and dermal cell types in wound healing processes. The method may involve adjusting culture conditions to modulate cell-cell adhesion between the first cells and second cells. In some implementations, the calcium concentration in the culture medium may be varied to control the strength of cell-cell adhesion. Lower calcium Princeton - 101476 concentrations may reduce adhesion, while higher concentrations may promote stronger cell- cell contacts. After creating the co-culture, the spatial organization and interactions between different cells may be analyzed through imaging techniques. In some cases, fluorescence microscopy may be used to visualize the distribution and arrangement of the different cell types within the patterned culture. Time-lapse imaging may also be employed to observe dynamic interactions between the cell populations over time. The first cultured cells and the second cultured cells may comprise different cell types beyond just keratinocytes and fibroblasts. In some implementations, the method may be used to create co-cultures with endothelial cells, smooth muscle cells, or various types of stem cells. This flexibility may allow researchers to study diverse cellular interactions relevant to different tissue types or disease models. FIG. 12 illustrates a schematic representation of a process for creating a co-culture using the automated scratching method. The figure shows a sequence of steps including scratching a pattern in the first cell type, adding a suspension of the second cell type, washing with PBS, and replacing the media. This process may be modified or expanded to incorporate additional cell types or patterning steps for more complex co-culture arrangements. In some cases, the co-culture system may be used to study the migration and invasion of one cell type into regions occupied by another cell type. For example, the method may be used to investigate how fibroblasts migrate into keratinocyte-populated areas in response to wound healing signals. The automated scratching apparatus may allow for the creation of precise geometric patterns in co-cultures. In some implementations, concentric rings or spiral patterns of alternating cell types may be generated to study how spatial arrangement influences cellular behavior and communication. In some cases, the co-culture method may be extended to create three-dimensional structures by layering multiple cell types. This approach may involve sequential scratching and cell deposition steps to build up complex tissue-like arrangements. The imaging and analysis of co-cultures may incorporate advanced techniques such as single- cell tracking or gene expression profiling to gain detailed insights into cell-cell interactions and signaling processes within the patterned cultures. Example. All of the key variables and challenges discussed here are the things at which a robot excels: precision, reproducibility, throughput / repetition, and programmability. Inspired by these Princeton - 101476 advantages, a low-cost robotic platform originally intended for art generation was modified. The modified device was called SCRATCH (scalable cellular resection apparatus to characterize healing). SCRATCH allows (1) complete programmability to produce almost any pattern, (2) the use of any scratching tip (e.g., pipette tips, needles, wires, etc.), (3) compatibility with nearly all standard culture vessels (e.g., 3.5 cm dishes to 96-well plates), (4) direct use ina sterile culture hood, and (5) a low net cost of ^ $500 at the time of writing. The remainderof this report summarizes how SCRATCH works and demonstrates its capabilities. SCRATCH is a fully automated scratch assay system, and its key advantages stem from computer control of a robotic gantry (see FIG. 1). The core of the SCRATCH device is a writing / drawing robot that provides programmable lateral (xy) and vertical (z) movement of the scratching apparatus (see xyz coordinate system in FIG.1). While SCRATCH can be built using off-the-shelf components from the 3D printing community, for simplicity, in this example, a hobby "art bot" (AxiDraw V3), originally intended to hold pens and markers, wasmodified to save time, and for a minimal cost ( ∼ $500 ). This chassis consists of an xy steppermotor-belt system to position a pipette tip tool over a tissue culture region and a servo motor to precisely and gently bring the tool into contact with the tissue in preparation for scratching. Instead of a pen or marker, a customized pipette tip holder for 10^^ L pipette tips was 3D printed, which could be tuned for any pipette tip style. To ensure stability of the tip during scratching, a thin layer of reusable adhesive putty was applied between the tip and the holder. This tip carrier can then be attached to the xyz gantry as if it were a pen. Here, the pipette holder assembly was then gently clamped to the vertical stage of the Axidraw using the built-in clamping screw. At this point, SCRATCH is ready for use. A key design goal was to make SCRATCH as user friendly and reproducible as possible to enable rapid adoption in cell biology labs, so a key feature of the example design is our modular sample holder directly attached to the frame of SCRATCH that allows most standard culture vessels—from 3.5-cm Petri dishes to 96-well plates to be positioned precisely and reproducibly relative to the pipette tool. Here, the culture vessels were press-fit into the modular plate holder. If needed, reusable adhesive could be added to improve stability. This sample holder also incorporates an alignment ring to calibrate the tip position at the beginning of the scratch. With the gantry in pen-up position and powered down (or its motors disengaged), the gantry arm was moved across the dish to ensure vertical clearance through the dish walls, and then Princeton - 101476 aligned the pipette tip with the mark on the alignment ring, thus establishing the ‘‘origin’’ of the drawing and the starting point. The use of the fixture allows SCRATCH to be controlled using pre-made template files in open-source drawing software (Inkscape already has plug-in support for many drawing bots). The user then loads an appropriate template for a given culture vessel, draws the desired patterns in each well, and ‘‘prints’’ the scratch pattern on SCRATCH via a connection (e.g., a USB connection. The versatility of SCRATCH was demonstrated by creating unique patterns in different types of Petri dishes and culture plates. First, a large-scale ‘‘star’’ pattern was created across a layer of primary mouse skin keratinocytes in a 35 mm dish (see FIGS. 3A-3B) to demonstrate the ability to generate complex, precise patterns (see FIG.3B). SCRATCH was then tested on a more challenging culture vessel: a 96-well plate. Here, the small well diameter prevents reproducible or precise manual scratching, and the throughput required to scratch 96 wells is not feasible using the traditional manual approach. However, SCRATCH was able to reliably pattern standard scratches in all 96 wells in <4 min; more complex geometries will take longer. Once calibrated, SCRATCH can automatically and reproducibly scratch arbitrary patterns in most standard culture dishes or plates at high throughput. It was first assessed how reproducible SCRATCH patterns were relative to manual patterns using linear scratches made in primary mouse skin keratinocyte layers cultured in 60 mm plates using E-medium (Nowak and Fuchs, 2009) supplemented with 15% serum (S11550, Atlanta Biologicals) and 50 mM calcium; representative results are shown in FIG.4. The standard deviation of the width of each scratch as the metric for evaluating uniformity. As shown in FIGS. 5A-5B, SCRATCH exhibited significantly improved uniformity vs. manualscratching (nearly 4 ൈ reduction in standard deviation and on the order of a single cell) whilemaintaining an average width of ~ 700 µm (approximate diameter of the 10 µL pipette tip). The observed variations we do see with SCRATCH likely reflect both biological variability in cell orientations and minor vibrations from the motor-belt system. In a separate test where a pen filled with protein-A was attached to the gantry, it was shown that the wobble in a straight line path in the x and / or y directions had a deviation of 20 µm, significantly less than the pipette tip width of 700 µm. Referring to FIG.14, in a separate test, it was also shown that SCRATCH (with the 10 µL pipette tip used in this example) was capable of generating scratches down to 1 mm spacing, but clearance was lost for scratches less than 1 mm apart. Princeton - 101476 Therefore, SCRATCH demonstrates superior uniformity to manual scratches in basic tissues, which improves reproducibility of scratch assays and allows higher throughput. As a demonstration of these benefits, an array of 15 linear gaps was rapidly produced into a primary mouse skin monolayer and the wound closure rate was quantified to validate the uniformity. See FIG.6. The closure curves indicate relatively uniform and tight healing dynamics. The importance of scratching speed (how quickly the tool is translated through the tissue) was investigated. This is something impossible to control manually, whereas SCRATCH allows scratch speed to be programmed up to 380 mm / s. Tissues are viscoelastic materials, meaning that their mechanical properties, adhesion to the substrate, and mechanobiological responses depend on the rate at which they are mechanically deformed, not just how much they are deformed, so being able to regulate the scratching rate should provide unique advantages and a new dimension to consider. In particular, it was hypothesized that the high-speed, precise motion of SCRATCH would be particularly useful when working with more challenging tissues possessing strong cell-cell adhesion and relatively weaker cell-substrate adhesion where slow or irregular manual scratching can cause the tissues to delaminate rather than being "cut." Here, the widespread MDCK (Madin-Darby canine kidney) kidney epithelial model was used, commonly used in all manner of collective migration experiments and screens and known to exhibit strong cell-cell adhesion and develop collective cell behaviors as a result. Wild-type MDCK-II cells were cultured in Dulbecco’s Modified Eagle’s Medium (D5523- 10L, Sigma-Aldrich) with 1 g / L sodium bicarbonate, 10% fetal bovine serum (S11550, Atlanta Biologicals), and 1% penicillin–streptomycin (15140-122, Gibco). A baseline was first established by manually scratching engineered, mature MDCK layers as best as could be accomplished, which resulted in massive, irregular gaps and widespread delamination due to inherent irregularities in the manual process. Similar results were observed when SCRATCH was set to a slow speed (38 mm / s) and the experiment was repeated. By contrast, when the experiment was repeated with SCRATCH set to the fastest translation speed (380 mm / s), it was able to produce highly uniform and more regular scratch patterns in comparison to slower mechanical or manual scratching (see FIG.7). Overall, SCRATCH was able to deliver more precision, reproducibility, and throughput than manual scratching. Subtractive tissue manufacturing: Designing complex tissue patterns. Only laboratory wounds are perfect straight lines, and many studies have emphasized the importance of tissue and wound shape in governing cellular migration and growth. This concept was explored by adapting SCRATCH for subtractive manufacturing of living tissues- gradually removing existing regions of tissue to produce complex patterns (returning to the Princeton - 101476 primary mouse skin monolayer model). SCRATCH enables this by "raster cutting," where it can gradually move the pipette tip tool back and forth while ensuring an overlap in the pattern to fully clear a given region of cells. See FIGS. 8 and 9A-9B. Here, an approximate overlap of 75% was chosen (e.g., for two passes through a given area, the scratch tip in the second pass will cover 75% of the area that the scratch tip covered in the first pass). However, it will be understood that overlap may be set to almost any degree (indeed, in some implementations, the tip may be configured to have at least one pass with 100% overlap of a previous pass, while in other implementations, the tip may be configured to have 1% overlap). Referring to FIGS.10A and 10B, "Positive" (10A) or "negative" (10B) patterns can be achieved by selectively scratching the "center" or "edge" of a monolayer, either leaving a solid tissue 1000 (positive) (1000) or cleared region 1002 (negative). This subtractive manufacturing method extends the application of SCRATCH beyond pure scratch assays to complex assays evaluating the role of wound size and shape, for example. Moreover, this process is also fully automated within the free software used to control SCRATCH, allowing arbitrarily complex patterns, as shown in FIGS.11A-11B. . SCRATCH for complex co-cultures. The "empty space" created by SCRATCH offers new potential for tissue co-culture because additional cell types can be back-filled into the newly created empty regions. See FIG.12. As a demonstration of this, a complex co-culture was created using a dermal / epidermal model of fibroblasts (NIH 3T3 fibroblasts) and keratinocytes (primary mouse keratinocytes). A 35mm dish with confluent keratinocytes was scratched with the steps shown previously. Then the dish was washed with PBS three times and stained with Cellbrite Green (30021, Biotium) at 5mL / mL for 30 min. A dish of 3T3 fibroblasts was also stained with Cellbrite Red (30023, Biotium) at 5mL / mL in suspension for 30 min. The stained dish is washed with PBS and 2mL co-culture media is added. Stained 3T3 suspension is washed with co-culture media 3 times using a centrifuge (5702, Eppendorf).3T3 suspension is then added to the keratinocyte dish with a density of 1000 cells / mm2. The dish is then incubated for 30 min for 3T3 attachment. Then the dish is fixed using 4% paraformaldehyde and stained with Hoechst 33342 (Thermo Fisher) for nucleus. The resulting spiral pattern is shown in FIGS.13A and 13B. The populations of keratinocytes 1300 and NIH 3T3 cells can be seen. The spiral is clearly visible, and the expanded view shows good spatial separation between keratinocytes and fibroblasts. Note that the quality of the backfilling method relies on the confluency of the first monolayer, since the seeded cells will also attach to the area that is outside of the intended region. In the case shown here, we did not Princeton - 101476 attempt to optimize the first tissue layer (e.g., by increasing cell density, allowing more culture time, or increase cell-cell junction strength with additional calcium), so some of the mesenchymal NIH 3T3 fibroblasts were able to "infiltrate" the keratinocyte layer (up to around 20% in the image shown). In practice, the co-culture conditions should be tuned for a desired application or question. Such are well understood in the art for any given co-culture. Similar to planar lithography, this process can be repeated multiple times for additional "layers" of cells as long as a co-culture medium exists that can support each cell type. These data further emphasize the versatility offered by the SCRATCH system to enable not only scratch assays but more complex tissue engineering and cell-cell communication assays. Thus, it can be seen that SCRATCH demonstrates a low cost, fully programmable, and high- throughput tool for the popular scratch assay that brings many significant advantages to the method, including improved reproducibility, throughput, and versatility with compatibility for nearly all standard culture plates and dishes. In particular, the disclosed technique has shown improved precision, throughput, and reproducibility over manual scratches as well as the ability to use scratching to produce unique tissue shapes and co-cultures without the need for microfabrication or manual stenciling. The open-source and open hardware nature of SCRATCH, combined with its low cost, should substantially aid its adoption, as it can be incorporated cheaply and easily into most cell biology laboratories and used in or outside of tissue culture hoods. A key aspect of SCRATCH is that it is easy to modify as a platform, allowing nearly any tip to be incorporated, and allowing for custom programming in Python if unique features are required that the standard graphics software does not allow (for instance, the tip can be programmed to go through a "wash" step where it is agitated in a buffer or ethanol well in between scratching different wells in a multiwell plate to avoid cross-contamination). Due to the potential for tip wear and cross- contamination, we strongly recommend replacing tips between different samples, but the user should evaluate the need for this in their own assays. Similarly, the SCRATCH platform can easily be modified with a more precise Z-drive to regulate scratching pressure or enable tip changes, and even the tips themselves can be adjusted or custom machined (e.g., from PTFE- Teflon) to reduce the risk of wear. Scratching inevitably leaves cell debris on the substrate, which may affect migration. However, compared to manual scratching, SCRATCH's programmable path allows the robot to make multiple passes, reducing cell debris attaching to the substrate. Moreover, SCRATCH is not dependent on one specific piece of hardware, as any traditional "maker" tools, such as a diode laser cutter or 3D printer, can be modified to do something Princeton - 101476 similar, and this concept is active area of research. Overall, this type of versatility can substantially improve the types of applications where scratch-style assays are useful and further aid in their adoption and accessibility to the broader community. SCRATCH is highly dependent on cell-cell adhesion due to its physical contact with the cells. A highly connective monolayer would result in greater width variation but, at the same time, would reduce "infiltration" of the second cell type for tissue co-culture. Carefully modulating tissue connectivity before and after scratching balances this tradeoff. Alternatively, non- contacting methods, such as laser ablation, can be used. Additionally, due to the conical shape of the pipette tips used in the example, areas close to the edge of the culture vessel cannot be reached. However, custom manufacturing of the scratching tips can alleviate the problem. A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims.

Claims

Princeton - 101476 CLAIMS 1. A device, comprising: a frame; a 3-axis gantry operably coupled to the frame; a scratch tip tool removably coupled to the 3-axis gantry; a sample holder operably coupled to the frame configured to receive a culture vessel containing cells; and at least one processing unit configured to: receive information comprising a target pattern; cause the scratch tip tool to scratch the cells in the target pattern.

2. The device of claim 1, wherein the 3-axis gantry is configured to have belt-driven XY axes and a servo or solenoid-driven Z-axis.

3. The device of claim 1, wherein the culture vessel is a Petri-style dish or a multiwell plate.

4. The device of claim 1, wherein the scratch tip tool comprises a 20 μL or smaller pipette.

5. The device of claim 1, wherein the scratch tip tool is removably coupled to a scratch tip tool holder.

6. The device of claim 5, wherein the scratch tip tool holder is removably coupled to the 3-axis gantry.

7. The device of claim 1, further comprising an alignment ring removably coupled to the laboratory dish or plate.

8. The device of claim 1, wherein the at least one processing unit is further configured to: control one or more motors of the 3-axis gantry to position the pipette tip tool; and adjust a scratching speed of the pipette tip tool based on properties of the cells.

9. The device of claim 8, wherein the at least one processing unit is further configured to control the motors to perform multiple passes of the pipette tip tool over a scratch area to ensure complete cell clearance.Princeton - 101476 10. The device of claim 9, wherein the at least one processing unit is further configured to generate a raster pattern for the scratch tip tool to create one or more geometric shapes in the cells.

11. A method for scratching a tissue, comprising: providing a device comprising a frame, a 3-axis gantry operably coupled to the frame, a scratch tip tool removably coupled to the 3-axis gantry, and a sample holder operably coupled to the frame; receiving, by at least one processing unit, information comprising a first target pattern; and causing, by the at least one processing unit, the scratch tip tool to scratch first cells in the first target pattern to form a first scratch pattern in the first cells, where the first cells are disposed in a culture vessel that is in contact with the sample holder.

12. The method of claim 11, further comprising establishing an origin by aligning a tip of the pipette tip tool with a target location on an alignment ring on the culture vessel.

13. The method of claim 12, further comprising attaching the alignment ring to the culture vessel prior to establishing the origin.

14. The method of claim 11, further comprising: washing the culture vessel with a buffered solution after scratching the first cells; adding a suspension comprising second cells to the culture vessel over the first cells; and forming a co-culture by incubating the culture vessel containing the first cells and the second cells, 15. The method of claim 14, wherein the first cultured cells and the second cultured cells comprise different cell types.

16. The method of claim 14, further comprising: scratching a second pattern in the co-culture; and adding third cells to the second scratched pattern.Princeton - 101476 17. The method of claim 14, wherein the first target pattern comprises a spiral, grid, or a geometric shape.

18. The method of claim 14, further comprising adjusting culture conditions to modulate cell- cell adhesion between the first cells and second cells.

19. The method of claim 14, further comprising imaging the co-culture to analyze spatial organization and interactions between different cells.

20. The method of claim 14, wherein the first cells comprise keratinocytes and the second cells comprises fibroblasts.

21. The method of claim 14, further comprising: staining the first cells prior to scratching; and staining the second cells prior to adding the suspension to the culture vessel.

22. The method of claim 21, further comprising fixing the culture vessel containing the first cells and the second cells after incubation.

23. The method of claim 11, wherein causing the scratch tip tool to scratch the first cells comprises: controlling one or more motors of the 3-axis gantry to position the scratch tip tool; and adjusting a scratching speed of the scratch tip tool based on properties of the first cells.

24. The method of claim 23, wherein causing the scratch tip tool to scratch the first cells further comprises controlling the motors to perform multiple passes of the pipette tip tool over a scratch area to ensure complete cell clearance.

25. The method of claim 24, wherein causing the scratch tip tool to scratch the first cells further comprises generating a raster pattern for the scratch tip tool to create one or more shapes in the first cells.

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