HIGH THROUGHPUT CELL MIGRATION ASSAY PLATES AND METHODS OF FABRICATION

Abstract

A Cell Migration Assay Plates (CMAP) assembly for high throughput microfluidic migration assays and method of manufacturing thereof are provided. The CMAP assembly includes a top plate having a plurality of wells aligned with a bottom plate having a plurality of troughs. Each of the plurality of wells is defined at least in part by first and second reservoirs and a divisional wall extending between the reservoirs. The bottom plate is secured to the top plate to form a plurality of micro-channels, such that each one of the plurality of micro-channels is defined by a portion of one of the divisional walls and a portion of a corresponding one of the plurality of troughs. The plurality of micro-channels enable communication between the reservoirs and visualization of cells migrating through the micro-channels. In this manner, migration of cells through the micro-channels can be visualized for testing and screening applications.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/104,093 filed on Oct. 22, 2020, the content of which is incorporated by reference in its entirety.

BACKGROUND

1. Field

The present inventive concept is directed to microfluidic devices for tumor cell migration with high throughput and fabrication methods thereof. The microfluidic devices can be used for tumor drug screening applications.

2. Discussion of Related Art

Conventional migration assays utilize two-dimensional (2D) surfaces for assessing tumor cell migration. In scratch/gap type assays, cells migrate towards an empty space via adhesion-dependent or mesenchymal-mode migration. In transwell assays, such as Boyden chamber, a dense extracellular matrix (ECM) and narrow pores are provided, and migration is driven by a chemotactic-gradient across a thin membrane. Neither the scratch/gap type assays nor the transwell assays are able to recapitulate any three-dimensional (3D) migration phenotypes of tumor cells seen in-vivo.

Although some more recent 3D assays, such as a multi-cellular tumor spheroid (MCTS), partially recreate the complex microenvironment of a tumor, such are not suitable for migration studies due to various reasons including that cells do not migrate within a spheroid of the tumor.

Further, commercial micro-channel devices are used in various biological applications. Conventional migration studies are conducted with polydimethylsiloxane (PDMS) based microfluidic devices fabricated using soft lithography. Although the PDMS-based migration devices are suitable for low throughput or proof-of-concept type of experiments, such are unsuitable for producing high density and high volume migration device arrays with reproducible channel dimensions, which are required for a high quality clinical assays.

Accordingly, there is a need to develop apparatuses and associated techniques for high throughput cell migration studies that do not suffer from the aforementioned deficiencies, are adaptable to accommodate a variety of different application requirements, and are efficient, economical, and easy to fabricate and utilize.

BRIEF SUMMARY

The present inventive concept provides multiple Cell Migration Assay Plates (CMAP) assemblies operable to function as high throughput microfluidic device plates for studying cell migration, methods to fabricate the microfluidic device plates and form the CMAP assemblies, and image analysis procedures to quantify cell migration using the CMAP assemblies.

The aforementioned may be achieved in one aspect of the present inventive concept by forming a CMAP assembly defined by a top plate and a bottom plate. The top plate has a plurality of wells arranged in an array with a plurality of columns and a plurality of rows. Each of the wells is defined at least in part by a pair of reservoirs. The bottom plate is a micro-fabricated plate having a plurality of troughs. When the top plate and the bottom plate are joined, a plurality of micro-channels are formed, which are defined by the plurality of troughs and a portion of the top plate. The micro-channels connect respective reservoirs in each of the pair of reservoirs. The top well plate and bottom plate can be manufactured with material and technologies compatible for large scale production with high feature reproducibility. In some embodiments, tumor cells can be seeded in one reservoir of the pair of reservoirs, referred to an input reservoir, while the other reservoir, referred to an output reservoir, is operable to receive the tumor cells after migration through one of the plurality of micro-channels. The micro-channels in the CMAP assembly can be designed to allow single cell migration and/or collective migration of tumor cells. The physical confinement of tumor cells in the micro-channels can trigger a 3D migration phenotype without any chemo-gradient between the two reservoirs. Migrating tumor cells are polarized due to spatial cues provided by the extended troughs and the micro-channels. Migrating cells have a polarity, e.g. a front and a back. Without the polarity, the cells would move in ail directions. With the spatially induced polarity, however, the cells are caused to move forward in the micro-channels. As such, dimensions (L×W×H) of troughs and micro-channels induce the 3D mode of migration and at the same time accomplish the migration assay in a reasonable time frame.

The CMAP assembly provides a 3D migration mode for tumor cells in the micro-channels. The 3D migration mode is similar to in-vivo migration mode. The 3D migration mode is fundamentally different than the 2D mesenchymal mode of migration. The difference in the mode of migration is important to evaluate drug response because the cytoskeleton of the cell undergoes a massive transformation, when a cell migrating on a 2D surface transitions to a 3D confined space. Additionally, the nucleus of the cell dramatically changes shape to facilitate movement in tightly-confined 3D space of the micro-channels. This change in the shape and size of the nucleus can modulate transcription and potentially affect cell cycle progression.

The CMAP assembly creates a physical environment, e.g., of a defined geometrical shape and size, to deliberately and controllably trigger a 3D migration phenotype observed in confined tumor cells. The CMAP assembly enables a user to study tumor cell migration, at a single cell level, without sacrificing any imaging resolution afforded by state-of-the-art microscopic imaging techniques. The CMAP assembly is operable to interrogate migration phenotype of tumor cells without any mechanical or chemical perturbation to cell culture. In contrast, conventional 2D surfaces used in conventional migration assays do not recapitulate 3D micro-environment, e.g., physically or bio-molecularly, of tumor tissue. The entire process of migration can be imaged and quantified in a high throughput plate reader using the CMAP assembly of the present inventive concept.

The disclosure provides different CMAP assemblies, e.g., a large array format and a small array format. In some embodiments, a small array format CMAP assembly, with an array four (4) sets of micro-wells arranged in a 2×2 pattern, has a same size as a standard microscope slide. The small array format CMAP assembly is designed for low throughput basic research applications to study 3D tumor cell migration.

In an embodiment, the large array format CMAP assembly, with an array of ninety-six (96) sets of micro-wells arranged in an 8×12 pattern, is dimensionally of a same size as an industry standard ninety-six (96) well plate for cell culture, with 240 micro-channels per well. One application of the CMAP assembly in the large array format is for high throughput screening of tumor drugs. The CMAP assembly in the large format array can have high throughput, high content imagers/plate readers, and can trigger in-vivo migration phenotype, which is necessary for a robust migration assay screening of a large library of drugs.

Additional aspects, advantages, and utilities of the present inventive concept will be set forth, in part, in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present inventive concept.

The foregoing is intended to be illustrative and is not meant in a limiting sense. Many features and subcombinations of the present inventive concept may be made and will be readily evident upon a study of the following specification and accompanying drawings comprising a part thereof. These features and subcombinations may be employed without reference to other features and subcombinations.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present inventive concept are illustrated by way of example in which like reference numerals indicate similar elements and in which:

FIG. 1A is a bottom perspective view of a top plate of a large format Cell Migration Assay Plates (CMAP) assembly showing ninety-six (96) wells extending through the top plate, prior to assembly of the large format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 1B is a top perspective view of a bottom plate of the large format CMAP assembly showing ninety-six (96) sets of troughs imprinted into the bottom plate, prior to assembly of the large format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 1C is a top perspective view of the top plate of FIG. 1A, in accordance with embodiments of the present inventive concept;

FIG. 1D is a top perspective view of the top plate and the bottom plate of FIGS. 1A-1C prior to assembly of the large format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 2A is a top plan view of a single well of the top plate of FIG. 1A showing two reservoirs prior to assembly of the large format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 2B is a top plan view of a single well of the top plate showing two reservoirs within the single well and a single set of troughs of the bottom plate assembled to form micro-channels, after assembly of the large format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 3A is a perspective cross-sectional view of the CMAP assembly showing micro-channels formed, upon assembly of the large format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 3B is a magnified view of FIG. 3A, with hidden portions illustrated, in accordance with embodiments of the present inventive concept;

FIG. 3C is an elevated side, cross-sectional view of the CMAP assembly showing micro-channels formed, upon assembly of the large format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 4A is a bottom perspective view of a top plate of a small format CMAP assembly showing four (4) wells with eight (8) reservoirs extending through the top plate, prior to assembly of the small format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 4B is a top perspective view of the top plate of FIG. 4A and a bottom plate showing four (4) sets of troughs, prior to assembly of the small format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 5 is a flow chart illustrating steps to form the large format CMAP assembly or the small format CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 6A is a top perspective view of the large format CMAP assembly with the top plate and the bottom plate of FIGS. 1A-1D being joined together via a welding process, in accordance with embodiments of the present inventive concept;

FIG. 6B is a top perspective view of the small format CMAP assembly with the top plate and the bottom plate of FIGS. 4A-4B being joined together via a welding process, in accordance with embodiments of the present inventive concept;

FIG. 7A is a magnified section view of FIG. 2B showing cells migrating through the micro-channels exemplary of a fluorescence image, in accordance with embodiments of the present inventive concept;

FIG. 7B is a magnified section view of FIG. 2B showing cells migrating through the micro-channels exemplary of a fluorescence image, in accordance with embodiments of the present inventive concept;

FIG. 8A is a Differential Interference Contrast (DIC) image of an input reservoir and an output reservoir and cells migrating through micro-channels of the CMAP assembly, in accordance with embodiments of the present inventive concept;

FIG. 8B is a DIC image showing cell migration in the micro-channels, in accordance with embodiments of the present inventive concept;

FIG. 8C is a magnified DIC image showing cell migration in the micro-channels, in accordance with embodiments of the present inventive concept; and

FIG. 8D is a magnified DIC image showing cell migration in the micro-channels, in accordance with embodiments of the present inventive concept.

The drawing figures do not limit the present inventive concept to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain embodiments of the present inventive concept.

DETAILED DESCRIPTION

The following detailed description references the accompanying drawings that illustrate various embodiments of the present inventive concept. The illustrations and description are intended to describe aspects and embodiments of the present inventive concept in sufficient detail to enable those skilled in the art to practice the present inventive concept. Other components can be utilized and changes can be made without departing from the scope of the present inventive concept. The following description is, therefore, not to be taken in a limiting sense. The scope of the present inventive concept is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled.

I. Terminology

The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present inventive concept or the appended claims.

Further, as the present inventive concept is susceptible to embodiments of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present inventive concept and not intended to limit the present inventive concept to the specific embodiments shown and described. Any one of the features of the present inventive concept may be used separately or in combination with any other feature. References to the terms “embodiment,” “embodiments,” and/or the like in the description mean that the feature and/or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “embodiment,” “embodiments,” and/or the like in the description do not necessarily refer to the same embodiment and are also not mutually exclusive unless so stated and/or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present inventive concept may include a variety of combinations and/or integrations of the embodiments described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present inventive concept will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present inventive concept, and be encompassed by the claims.

Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

The terms “comprising,” “including” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including” and “having” mean to include, but not necessarily be limited to the things so described.

Lastly, the terms “or” and “and/or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and/or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

II. General Architecture

Turning to FIGS. 1A-1D, a large format Cell Migration Assay Plates (CMAP) assembly 100 is illustrated according to an embodiment of the present inventive concept. The CMAP assembly 100 is generally defined by a top plate 102 and a bottom plate 104.

The top plate 102 includes ninety-six (96) wells 106 extending entirely through the top plate 102. The wells 106 are arranged in an array 108, with eight (8) rows 110 and twelve (12) columns 112. The top plate 102 may also optionally include chamfered corners on one side of the top plate 102. The chamfered corners can serve as alignment markers to facilitate assembly of a cover of a similar shape, e.g., with corresponding chamfered corners, onto the top plate 102 to close one side of the wells 106.

Each of the wells 106 is defined by a perimeter sidewall 114 with a top peripheral edge 116 spaced from a bottom peripheral edge 118 of the perimeter sidewall 114. The perimeter sidewall 114 defines a set of reservoirs 120, e.g., a first reservoir and a second reservoir extending entirely through the top plate 102, thereby causing each of the wells 106 to be substantially bottomless.

The bottom plate 104 includes ninety-six (96) trough sets 122 formed into a substantially planar surface 124 on one side 126 of the bottom plate 104. Each set of the troughs sets 122 includes a linear array of troughs 128 that corresponds to one of the wells 106. Each trough of the linear array of troughs 128 is defined by opposing sidewalls 130, 132, a bottom wall 134 extending between the sidewalls 130, 132, and opposing end walls 136, 138 extending between the sidewalls 130, 132. The opposing sidewalls 130, 132, the bottom wall 134, and the end walls 136, 138 collectively define an elongated cavity 140 for receiving one or more cells or drugs.

It is foreseen that the linear array of troughs 128 may include any number of troughs, with different ones of the troughs being of different shapes and/or sizes, without deviating from the scope of the present inventive concept. Indeed, the number, size, and shape of troughs shown by the figures are merely for illustrative purposes for understanding the present inventive concept. In an embodiment, the linear array of troughs 128 includes two-hundred and forty (240) troughs, with a cross-section of 5 μm×5 μm, and a length of 700 μm.

As illustrated, FIGS. 1A and 10 respectively show bottom and top perspective views of the top plate 102 of the large format CMAP assembly 100 prior to assembly to the bottom plate 104, which is illustrated via FIG. 1B. FIG. 1D is a top perspective view of the top plate 102 aligned with the bottom plate 104 prior to securing the top plate 102 to the bottom plate 10 to form the large format CMAP assembly 100.

Turning to FIG. 2A, a magnified top plan view of one of the wells 106 with the set of reservoirs 120 of the top plate 102 is illustrated, prior to assembly of the large format CMAP assembly 100. Each of the reservoirs 120 includes the perimeter sidewall 114 with the top peripheral edge 116 spaced from the bottom peripheral edge 118 of the perimeter sidewall 114. A separation or divisional wall portion 204 extends between the reservoirs 120. In an embodiment, the divisional wall portion 204 separates the reservoirs 120 with a thickness of 500 μm. It is foreseen that the thickness of the divisional wall portion 204 may be greater or smaller without deviating from the scope of the present inventive concept.

Turning to FIG. 2B-3C, one of the wells 106 with the set of reservoirs 120 of the top plate 102 and one of the linear arrays of troughs 128 of the bottom plate 104 are illustrated, after assembly of the large format CMAP assembly 100. When the top plate 102 and the bottom plate 104 are assembled to form the large format CMAP assembly 100, the planar surface 124 of the bottom plate 104 abuts the bottom peripheral edge 118 of the perimeter sidewall 114 such that each reservoir of the set of reservoirs 120 and each of the wells 106 are sealed by the bottom plate 104 and micro-channels 210 are formed, which fluidly connect each reservoir of the set of reservoirs 120.

Each of the micro-channels 210 includes an entrance opening 212 and an exit opening 214 at opposite ends thereof to define a one-way direction of fluid communication between each reservoir of the set of reservoirs 120. Each of the micro-channels 210 is defined by a middle portion 220 of each trough of the linear array of troughs 128 and a surface of the divisional wall portion 204, which functions as a micro-channel roof. On one side of the middle portion 220, an entry portion 224 of each trough of the linear array of troughs 128 does not have the micro-channel roof and, therefore, remains open into a first one of the reservoirs 120, which is operable to function as a seeding or input reservoir. In this manner, the input reservoir can be used to temporarily contain a cell and guide the cell into a respective one of the micro-channels 210. On another side of the middle portion 220, an exit portion 226 of each trough of the linear array of troughs 128 also does not have the micro-channel roof and, therefore, also remains open into a second one of the reservoirs 120, which is operable to function as an output reservoir. In this manner, the output reservoir can be used to receive the tumor cells after migration through one of the plurality of micro-channels. The entry portion 224 and the exit portion 226 advantageously provide an extra margin in case of misalignment between the linear array of troughs 128 and the divisional wall portion 204 during assembly of the top plate 102 and the bottom plate 104. In an embodiment, when properly aligned, each trough of the linear array of troughs 128 has a length of approximately 700 μm, and the entry portion 224 and the exit portion 226 respectively protrude approximately 100 μm past either side of the divisional wall 204, which has a thickness of approximately 500 μm. In an embodiment, the micro-channels 210 have varying lengths and/or cross-sections. For instance, it is foreseen that a width of the divisional wall 204 may be increased or decreased to respectively increase or decrease a length and/or cross-section of the micro-channels 210. In this manner, the top plate 102, and the bottom plate 104 are advantageously operable to function as microfluidic device plates that enable a user to interrogate migratory potential of cells such as tumor cells.

In an embodiment, the large format CMAP assembly 100 includes 270×96 or 25,920 of the micro-channels 210. In an embodiment, the micro-channels 308 may have a square cross-section, e.g. 5 μm×5 μm, or a rectangular cross-section, e.g. 5 μm by 3 μm. It is foreseen that the number of micro-channels 210 may be of greater number or smaller number and/or of greater size or of smaller size, without deviating from the scope of the present inventive concept. In this manner, given the high number of micro-channels 210, the CMAP assembly 100 is advantageously operable to provide a high throughput relative to a basic microfluidic migration device.

As illustrated, FIG. 3A shows a perspective view of the CMAP assembly 100 with the micro-channels 210 formed, FIG. 3B shows a magnified view of FIG. 3A, except with portions hidden by the divisional wall portion 204 illustrated, and FIG. 3C shows a side view of the micro-channels 210 formed via the top plate 102 and the bottom plate 104.

Turning to FIGS. 4A-B, a small format Cell Migration Assay Plates (CMAP) assembly 400 is illustrated according to an embodiment of the present inventive concept. The CMAP assembly 400 is generally defined by a top plate 402 and a bottom plate 404.

The top plate 202 includes four (4) wells 406 extending entirely through the top plate 202. The wells 406 are arranged in an array 408 with two (2) rows 411 and two (2) columns 412. Each of the wells 406 is defined by a perimeter sidewall 414 with a top peripheral edge 416 spaced from a bottom peripheral edge 418 of the perimeter sidewall 414. The perimeter sidewall 414 defines a set of reservoirs 420, e.g., a first reservoir and a second reservoir, extending entirely through the top plate 202, thereby causing each of the wells 406 to be substantially bottomless.

The bottom plate 404 includes four (4) trough sets 422 formed into a planar surface 424 on one side 426 of the bottom plate 404. Each set of the troughs sets 422 includes a linear array of troughs 428 that correspond to a respective one of the wells 406. Similar to each trough of the linear array of troughs 128, each trough of the linear array of troughs 428 is defined by opposing sidewalls, a bottom wall extending between the sidewalls, and opposing end walls extending between the sidewalls. The opposing sidewalls, the bottom wall, and the end walls collectively define an elongated cavity for receiving one or more cells or drugs.

It is foreseen that the linear array of troughs 428 may include any number of troughs, with different ones of the troughs being of different shapes and/or sizes, without deviating from the scope of the present inventive concept. Indeed, the number, size, and shape of troughs shown by the figures are merely for illustrative purposes for understanding the present inventive concept. In an embodiment, the linear array of troughs 428 includes two-hundred and forty (240) troughs, with a cross-section of 5 μm×5 μm, and a length of 700 μm.

Each reservoir of the set of reservoirs 420 includes the perimeter sidewall 414 extending through the bottom plate 404. The top plate 402 includes a separation or divisional wall portion 444 extending between the set of reservoirs 420. In an embodiment, the divisional wall portion 444 separates the set of reservoirs 420 with a thickness of 500 μm. It is foreseen that the thickness of the divisional wall portion 444 may be greater or smaller without deviating from the scope of the present inventive concept.

When the top plate 402 and the bottom plate 404 are assembled to form the small format CMAP assembly 400, the planar surface 424 of the bottom plate 404 abuts the top plate 402 such that each reservoir of the set of reservoirs 420 and each of the wells 406 are sealed by the bottom plate 404 and micro-channels are formed, which fluidly connect each reservoir of the set of reservoirs 420.

Similar to the micro-channels 210, each of the micro-channels 410 include an entrance opening and an exit opening at opposite ends thereof to define a one-way direction of fluid communication between each reservoir of the set of reservoirs 420. Each of the micro-channels 410 are defined by a middle portion of each trough of the linear array of troughs 428 and a surface of the divisional wall portion 444, which functions as a micro-channel roof. On one side of the middle portion, an entry portion of each trough of the linear array of troughs 428 does not have the micro-channel roof and, therefore, remains open into a first one of the set of reservoirs 420, which is operable to function as a seeding or input reservoir. In this manner, the input reservoir can be used to temporarily contain a cell and guide the cell into a respective one of the micro-channels 410. On another side of the middle portion, an exit portion of each trough of the linear array of troughs 428 also does not have the micro-channel roof and, therefore, also remains open into a second one of the set of reservoirs 420, which is operable to function as an output reservoir. In this manner, the output reservoir can be used to receive the tumor cells after migration through one of the plurality of micro-channels. The entry portion and the exit portion advantageously provide an extra margin in case of misalignment between the linear array of troughs 428 and the divisional wall portion 444 during assembly of the top plate 402 and the bottom plate 404. In an embodiment, when properly aligned, each trough of the linear array of troughs 428 has a length of approximately 700 μm, and the entry portion and the exit portion respectively protrude approximately 100 μm past either side of the divisional wall portion 444, which has a thickness of approximately 500 μm. In an embodiment, the micro-channels 410 have varying lengths and/or cross-sections. For instance, it is foreseen that a width of the divisional wall portion 444 may be increased or decreased to respectively increase or decrease a length and/or cross-section of the micro-channels 410. In this manner, the top plate 402, and the bottom plate 404 are advantageously operable to function as microfluidic device plates that enable a user to interrogate migratory potential of cells such as tumor cells.

As illustrated, FIGS. 4A and 4B respectively show bottom and top perspective views of the top plate 402 of the small format CMAP assembly 400 prior to assembly to the bottom plate 404.

Micro-Channel Design

The CMAP assemblies 100, 400 can be designed with the micro-channels 210, 410 of various geometries, such as varying lengths, varying widths, and/or varying heights, which advantageously allow testing of and experimentations with various types of tumor cells and/or drugs.

The micro-channel dimensions (e.g. cross-section and length) can be selected for a single cell migration assay or a collective cell migration assay. When one of the micro-channels 210, 410 is wide and provides less physical confinement for cells, the one of the micro-channels 210, 410 provides two-dimensional (2D) migration for the cells and the cells do not touch fewer than all, e.g., one or two surfaces of the sidewalls 130, 132, the bottom wall 134, and the surface of the divisional wall portion 204, 444, which functions as the micro-channel roof. Conversely, when one of the micro-channels 210, 410 is narrow and provides greater physical confinement for cells, the cells are forced to squeeze through the one of the micro-channels 210, 410, for example, by touching all surfaces of the sidewalls 130, 132, the bottom wall 134, and the surface of the divisional wall portion 204, 444, which functions as the micro-channel roof. In this manner, greater physical confinement of the one of the micro-channels 210, 410 provides three-dimensional (3D) migration for cells.

The migration time of a cell in the micro-channels 210, 410 may vary between different ones of the micro-channels 210, 410 with different cross-section areas. For instance, if a same cell is caused to pass through a first one of the micro-channels 210, 410 and a second one of the micro-channels 210, 410, the cell may take more time to pass through the first one of the micro-channels 210, 410 than the second one of the micro-channels 210, 410 if the first one of the micro-channels 210, 410 has a smaller cross-section area than the second one of the micro-channels 210, 410. In other words, cells move easier through the micro-channels 210, 410 with larger cross-section areas than through the micro-channels 210, 410 with smaller cross-sections.

In some embodiments, the micro-channels 210, 410 may have cross-section areas of a square shape, a rectangular shape, and/or a circular shape. Additionally, in some embodiments, the micro-channels 210, 410 may have a constant or consistent cross-section area. Additionally, in some embodiments, the micro-channels 210, 410 may have varying aspect ratios (e.g. a ratio of height to width) or varying heights and/or varying widths, for example, cross-section areas may vary along the length of a single one of the micro-channels 210, 410. For example, the single micro-channel may start with a width of 20 μm, then gradually contract to a width of 15 μm, a width of 10 μm, and a width of 5 μm. With such a varying cross-section area, the single micro-channel is advantageously operable to test cells in multiple one of the micro-channels 210, 410, e.g. four micro-channels, having widths of 20 μm, 15 μm, 10 μm, and 5 μm, respectively.

Additionally, in some embodiments, the cross-sections of the micro-channels 210, 410 may continuously decrease or at discrete steps, and/or may continuously increase or at discrete steps. Additionally, in some embodiments, the dimensions of the micro-channels 210, 410 may vary at discrete steps, for example, from width A, width B, width C, and width D, etc. For example, widths A, B, C, and D may decrease, or increase sequentially, or may vary with any pattern. Note that the physical gradient of the micro-channels 210, 410 are different from the chemo-gradient. There is no chemo-gradient between the set of reservoirs 120, 420, e.g. the input reservoir and the output reservoir.

In some embodiments, the micro-channels 210, 410 may have a cross-section ranging from 3 by 3 μm² to 20 by 20 μm². In some embodiments, the micro-channels 210, 410 may have a cross-section 5 by 5 μm². In some embodiments, the micro-channels 210, 410 may have a cross-section 10 by 10 μm². In some embodiments, the micro-channels 210, 410 may have a cross-section 15 by 15 μm². In some embodiments, the micro-channels 210, 410 may have a cross-section 3 by 5 μm². In some embodiments, the micro-channels 210, 410 may have a cross-section 3 by 10 μm². In some embodiments, the micro-channels 210, 410 may have a cross-section 5 by 10 μm². In some embodiments, the micro-channels 210, 410 may have a cross-section 5 by 15 μm². In some embodiments, the micro-channels 210, 410 may have varying lengths, for example, the length of the micro-channels 210, 410 may vary from 100 μm to 2 mm long. One of the micro-channels 210, 410 may have a different length from another one of the micro-channels 210, 410.

In some embodiments, the micro-channels 210, 410 may have a length ranging from 100 μm to 2.0 mm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 100 μm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 200 μm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 300 μm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 400 μm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 500 μm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 600 μm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 700 μm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 800 μm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 900 μm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 1.0 mm. In some embodiments, the micro-channels 210, 410 may have a length equal to or greater than 1.5 mm.

In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 2.0 mm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 1.5 mm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 1.0 mm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 900 μm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 800 μm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 700 μm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 600 μm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 500 μm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 400 μm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 300 μm. In some embodiments, the micro-channels 210, 410 may have a length less than or equal to 200 μm.

A higher density of the micro-channels 210, 410 is preferable for higher throughput applications of the present inventive concept. In some embodiments, the micro-channels 210, 410 may form an array including 50 to 400 micro-channels. In some embodiments, the micro-channels 210, 410 may form an array including 50 or more of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 100 or more of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 150 or more of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 200 or more of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 250 or more of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 300 or more of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 350 or more of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 240 micro-channels.

In some embodiments, the micro-channels 210, 410 may form an array including 400 or fewer of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 350 or fewer of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 300 or fewer of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 250 or fewer of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 200 or fewer of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 150 or fewer of the micro-channels 210, 410. In some embodiments, the micro-channels 210, 410 may form an array including 100 or fewer of the micro-channels 210, 410.

It will be appreciated by those skilled in the art that the dimensions, shape, and/or number of the micro-channels 210, 410 may vary with applications.

In some embodiments, of the micro-channels 210, 410 can be coated with extracellular matrix (ECM) molecules and/or filled with cells to create a tumor tissue like micro-environment.

One of the benefits of the CMAP assemblies 100, 400 is the high density of the micro-channels 210, 410, e.g., 240 micro-channels in each well, in an industry standard format, e.g., ninety-six (96) wells, and a total 23,040 micro-channels per plate.

Fabrication of CMAP Assembly

A fabrication process 500 includes forming the top plate 102, 402 using cyclic olefin polymer (COP) of a black color. In this manner, laser welding of the top plate 102, 402 can be facilitated and artifact-free imaging due to reduced reflection and fluorescence can be obtained via the black COP. Using the black COP, the top plate 102, 402 is formed using an injection molding process, at operation 502. Injection molding is a manufacturing process for producing plastic parts by injecting molten material into a mold. For example, the mold may have the pattern of the top plate 102, 402, such as including the wells 106 and the reservoirs 120. COP pellets are fed into a heated barrel and injected into a mold cavity, where the COP cools down to form the top plate 102, 402.

Next, the fabrication process 500 includes forming the bottom plate 104, 404 using an optically clear COP, which provides an optically clear window when viewing cell migration through the micro-channels 210, 410 of the bottom plate 104, 404. In this manner, the bottom plate 104, 404 enables inspection or imaging of cells migrating through the micro-channels 210, 410 and analysis of images to visualize cell migrations. Using the optically clear COP, the bottom plate 104, 404 is formed using a hot embossing process, at operation 504. The hot embossing process, which is a process of structuring polymer films or sheets by pressing a stamp into the polymer while the polymer is heated about its glass transition temperature. The polymer is much thicker than the height of the stamp structures. The relief is a perturbation of the total thickness of the polymer. Hot embossing is less prone to defects than nanoimprint lithography and is not limited to nano-structures or micro-structures. First, a silicon master mold is created with the imprint of the desired trough sets 122, 422. SU-8 is a commonly-used epoxy-based negative photoresist. Each set of the trough sets 122, 422 includes the linear array of troughs 128, 428, e.g. two-hundred and forty (240) troughs. The silicon SU-8 master mold is produced using a lithography process of spin coating SU-8 photoresist on a silicon wafer and then by exposure to UV light in a mask aligner. Then, non-cross-linked photoresist is washed away. The silicone SU-8 master mold with features of each trough in the linear array of troughs 128, 428 is produced. Next, negative stamps of silicone are produced by pouring and peeling silicone on silicon mater. In the final step, the bottom plate 104, 404 is fabricated by transferring or imprinting features on the silicone stamp onto plain sheets of COP by the hot embossing process. In some embodiments, the COP sheets may have a thickness ranging from 100 μm to 800 μm. In some embodiments, the COP sheets may have a thickness ranging from 100 μm to 400 μm. In some embodiments, the COP sheets may be 188 μm thick. In this manner, the bottom plate 104, 404 is micro-fabricated.

Next, the fabrication process 500 includes aligning the top plate 102, 402 and the bottom plate 104, 404, at operation 506. Once aligned, the top plate 102, 402 and the bottom plate 104, 404 are held by fixture and vacuum. For example, the top plate 102, 402 and the bottom plate 104, 404 can be aligned for precision positioning of the trough sets 122, 422 with respect to the divisional wall portions 204, 444, and then held under suction, via a vacuum, on a 3D automated translation stage system during a joining process, at operation 508.

The joining process includes joining or bonding the top plate 102, 402 and the bottom plate 104, 404 together to form the CMAP assembly 100, 400. The top plate 102, 402 and the bottom plate 104, 404 are joined or bonded together and fluidly sealed by a laser welding process at an interface or junction between the top plate 102, 402 and the bottom plate 104, 404 along peripheries of the set of reservoirs 120, 420 and the wells 106, 406, as further discussed hereafter.

Laser welding provides a number of advantages over other bonding processes such as gluing. Laser welding is faster than gluing and can also be used for automatic and large scale processes. Further, bonding provided via glue generally has low throughput, and potential complications due to chemical interactions with cells. The laser welding process of the present inventive concept is automatic and faster, and therefore has higher throughput. Further, the laser welding process of the present inventive concept better ensures no leaks will form and does not require chemicals. Other attachment processes such as via a friction-fit engagement and/or gluing provides a number of advantages over welding. For instance, the top plate 102, 402 and the bottom plate 104, 404 can be joined together to form the CMAP assembly 100, 400 so that the top plate 102, 402 is selectively detachable from the bottom plate 104, 404. For example, the joining process may utilize a friction-fit engagement and/or a reusable adhesive to allow the user to selectively attach and detach the top plate 102, 402 from the bottom plate 104, 404. In this manner, the user is advantageously provided with direct access to the cells migrating through the micro-channels 210, 410, thereby allowing further testing and/or inspection.

Turning to FIG. 6A, the operation 506 to form the CMAP assembly 100 is illustrated. The top plate 102 and the bottom plate 104 are aligned and held under suction on a 3D automated translation stage system including translation stages during the laser welding process. Next, via the operation 508, a laser spot from a laser source 602 is focused on a first interface between the top plate 102 and the bottom plate 104. Using alignment markers as a reference on the bottom plate 104, the translation stages move in a fixed 2D pattern, with the laser source 602 activated, thereby causing the bottom plate 104 to be welded, joined, and secured to the top plate 102 along each periphery of the wells 106, in a square pattern. In this manner, the top plate 102 and the bottom plate 104 are fluidly sealed to each other, and the CMAP assembly 100, with the micro-channels 210, is formed.

Turning to FIG. 6B, the operation 506 to form the CMAP assembly 400 is illustrated. The top plate 402 and the bottom plate 404 are aligned and held under suction on a 3D automated translation stage system including translation stages during the laser welding process. Next, via the operation 508, a laser spot from a laser source 652 is focused on a first interface between the top plate 402 and the bottom plate 404. Using alignment markers as a reference on the bottom plate 404, the translation stages move in a fixed 2D pattern, with the laser source 652 activated, thereby causing the bottom plate 404 to be welded to the top plate 402 along each periphery of the wells 406, in a square pattern. Next, the laser spot from the laser source 652 is focused on a second interface between the top plate 402 and the bottom plate 404. Using alignment markers as a reference on the bottom plate 404, the translation stages move in another fixed 2D pattern with respect to the laser spot, thereby causing the bottom plate 404 to be further welded to the top plate 402 along each of the reservoir peripheries, in a square pattern. In this manner, the top plate 402 and the bottom plate 404 are fluidly sealed to each other, and the CMAP assembly 400, with the micro-channels 410, is formed.

Image Analysis for Visualization of Cell Migrations

The present inventive concept utilizes a Fluorescence, Bright Field, plus Differential Interference Contrast (DIC) microscopy. DIC is an optical microscopy imaging method based on the principle of optical interference. Polarized light is split into two beams before it illuminates the sample and combined after exiting it. The combined beams form an interference image which maps the optical thickness of the sample.

Turning to FIGS. 7A and 7B, cells in different stages of migration through different ones of the micro-channels 210 are illustrated, which are exemplary of fluorescence images. FIG. 7A illustrates the CMAP assembly 100, with a constant aspect ratio at various times in accordance with embodiments of the present inventive concept. FIG. 7A shows that cells move along the micro-channels 210, to different positions 702, 704, 706, and 708 at 0 hours, 3 hours, 6 hours, and 9 hours, respectively, from left to right, in the micro-channels 210, with fixed dimensions. These cells may be put into an input reservoir of the reservoirs 120 and enter the entry portion 224 of one or more troughs, at different times. In an embodiment, straight ones of the micro-channels 210 are 5(w)×5(h) or 5×10 μm. In an embodiment, segments of tapered ones of the micro-channels 210 are 20(w)×10(h), 15×10 μm, 10×10 μm, 8×10 μm and 5×10 μm, respectively. For example, more cells may be added at 12 hours, 24 hours, and 36 hours after the first cell was added to the input reservoir.

FIG. 7B illustrates the CMAP assembly 100, with a varying aspect ratio at various times in accordance with embodiments of the present inventive concept. FIG. 7B shows that cells move along the micro-channels 210, to different positions 712, 714, 716, 718, 720, and 722 from left to right at 0 hours, 4 hours, 16 hours, 18 hours, 22 hours, and 25 hours, respectively, in the micro-channels 210, with varying dimensions. In an embodiment, straight ones of the micro-channels 210 are 5(w)×5(h) or 5×10 μm. In an embodiment, segments of tapered ones of the micro-channels 210 are 20(w)×10(h), 15×10 μm, 10×10 μm, 8×10 μm and 5×10 μm, respectively. Again, these cells may be put in the input reservoir of the reservoirs 120, and enter the entry portion 224 of one or more troughs, at different times. For example, more cells may be added at 12 hours, 24 hours, and 36 hours after the first cell was added to the input reservoir. It will be appreciated, by those skilled in the art, that the cells can be added to the input reservoir at any desired pattern, e.g., at different times, for testing.

Turning to FIGS. 8A-8D, fluorescence plus DIC images of DAPI-stained cells in different stages of migration through the micro-channels 210 are shown. DAPI or 4′, 6-diamidino-2-phenylindole is a fluorescent stain used to improve visualization and imaging of cells with the CMAP assembly, 100, 400. FIG. 8A shows a cell body 908, which has moved from an input reservoir 802 of the reservoirs 120 to a position along one of the micro-channels 210. FIG. 8B shows a cell body 810, which has moved from the input reservoir 802 to a position along one of the micro-channels 210. FIG. 8C shows a magnified image of a cell body 812, which has moved to a position along one of the micro-channels 210. FIG. 8D shows five (5) cells 814 A-E, which have moved to various positions along a single one of the micro-channels 210. In this manner, migration of cells through the micro-channels 210 can be visualized. With this visualization, cell migration can be studied for various purposes such as screening various tumor drugs.

Applications

Drug development is a time-consuming and prohibitively-expensive process. High failure rates of tumor drugs can be attributed, in part, to poor selectivity of drug molecules during in-vitro screening. The CMAP assembly 100, 400 significantly improves in-vitro drug screening sensitivity of tumor drugs.

The CMAP assembly 100, 400 facilitates drug screening applications where high throughput and high content capability are beneficial, for example, when a large library of drug molecules need to be screened. The CMAP assembly 100 with ninety-six (96) wells 106 is particularly designed to provide an optically clear bottom plate or window, enable high throughput, and be compatible with high content plate imagers (e.g. DIC microscopy) for drug screening assays.

Cancer or tumor cells generally have a big nucleus, with a width varying from 5 μm to 50 μm. Cancer cells may also be stiffer than normal cells. Cancer cells may squeeze through and migrate through the micro-channels 210, 410. However, an effective drug may block or stop the cancer or tumor cells from migrating through the micro-channels 210, 410. Indeed, various types of drugs can be tested with the cancer cells using the micro-channels 210, 410. If the drug prevents the cancer cells from migrating through the micro-channels 210, 410, such is indicative that the drug can cure cancer.

In some embodiments, cell culture protocols are provided. For instance, cell culture protocol may vary based on a number of factors including type of cells and/or type of assay. Or, cell culture protocol may be a single, generic cell culture protocol applicable regardless of any factors such as, but not limited to cell type and/or assay type. Cells can be cultured with drugs prior to drug testing in the CMAP assembly 100, 400. For example, with drug X tested along with cancer or tumor cells, the cells may only migrate half of a distance through the micro-channels 210, 410 relative to the cells' migration through the micro-channels 210, 410 without use of the drug X. With drug Y tested along with cancer or tumor cells, the cells may move a quarter of the distance through the micro-channels 210, 410 relative to the cells' migration through the micro-channels 210, 410 without use of the drug Y. As such, these tests demonstrate that the drug Y is more effective than the drug X to block movement of cancer or tumor cells within the micro-channels 210, 410. Therefore, drug Y may be more effective than drug X to cure cancer.

A drug may work well at the beginning to block cell migration through the micro-channels 210, 410, but may not work well later. As discussed herein, dimensions of a single one of the micro-channels 210, 410 can be designed to have reduced dimensions, for example, starting with a cross-section area of 20 by 20 μm², which continually reduces to a cross-section area of 15 by 15 μm², then to a cross-section area of 10 by 10 μm², and finally to a cross-section area of 5 by 5 μm². As such, the single one of the micro-channels 210, 410 is advantageously operable to yield a cell migration study in what would otherwise require four different ones of the micro-channels 210, 410.

The CMAP assembly 100, 400 have been used to test patient-derived glioma cells. In addition, migration tests with lung cancer cells and breast cancer cells have been conducted using the CMAP assembly 100, 400.

In some embodiments, the CMAP assembly 100, 400 can also be used as a diagnostic assay to score tumor invasion potential of individual patients. For example, different tumor cells may be tested. Tumor cells that demonstrate higher or quicker migration through the micro-channels 210, 410 of the CMAP assembly 100, 400 may be concluded to be more invasive to humans than tumor cells with lower or slower migration through the micro-channels 210, 410 of the CMAP assembly 100, 400.

Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present inventive concept. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present inventive concept. Accordingly, this description should not be taken as limiting the scope of the present inventive concept.

Those skilled in the art will appreciate that the presently disclosed embodiments teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall therebetween.

Claims

1. A cell migration assay plates (CMAP) assembly comprising:

a top plate having a plurality of wells, each of the plurality of wells defined at least in part by a first reservoir, a second reservoir, and a divisional wall extending between the first reservoir and the second reservoir;

a bottom plate operable to be secured to the top plate, the bottom plate having a plurality of troughs; and

a plurality of micro-channels formed when the bottom plate is secured to the top plate, each of the plurality of micro-channels defined by a portion of the divisional wall of each of the plurality of wells and middle portions of the plurality of troughs, the plurality of micro-channels enabling communication between the first reservoir and the second reservoir when the bottom plate is secured to the top plate.

2. The CMAP assembly of claim 1,

wherein, each of the middle portions of the plurality of troughs is connected to an entry portion and an exit portion, the entry portion and the exit portion extend away from the divisional wall on opposite sides of the divisional wall, when the bottom plate is secured to the top plate.

3. The CMAP assembly of claim 1,

wherein, the first reservoir and the second reservoir are elongated reservoirs, and the first reservoir extends parallel to the second reservoir.

4. The CMAP assembly of claim 1,

wherein, the plurality of troughs includes a plurality of trough sets, and each of the plurality of trough sets includes a first linear array of troughs and a second linear array of troughs.

5. The CMAP assembly of claim 4,

wherein, each of the plurality of troughs sets extend between a respective one of the plurality of wells.

6. The assembly of claim 1,

wherein, the communication is one-way communication from the first reservoir to the second reservoir, the first reservoir is a seeding reservoir for tumor cells and/or one or more drugs in each of the plurality of wells, and the second reservoir is an output reservoir to which the tumor cells or the one or more drugs are operable to migrate via a three-dimensional (3D) migration without application of any gradient between the first reservoir and the second reservoir in response being physical confined by the plurality of micro-channels.

7. The assembly of claim 1, wherein the plurality of wells include four (4) wells or ninety-six (96) wells.

8. The assembly of claim 1, wherein each of the plurality of micro-channels include two-hundred and forty (240) micro-channels.

9. The assembly of claim 1, wherein the plurality of micro-channels have varying lengths.

10. The assembly of claim 1, wherein lengths of the plurality of micro-channels vary from 100 μm to 2.0 mm.

11. The assembly of claim 1, wherein the plurality of micro-channels have varying aspect ratios of height-to-width.

12. The assembly of claim 1, wherein the plurality of micro-channels have dimensions decreasing continuously.

13. The assembly of claim 1, wherein the plurality of micro-channels have dimensions reduced at discrete steps.

14. The assembly of claim 1, wherein the plurality of micro-channels have dimensions increasing continuously.

15. The assembly of claim 1, wherein the plurality of micro-channels have dimensions increased at discrete steps.

16. The assembly of claim 1, wherein the divisional wall has a thickness ranging from 100 μm to 400 μm.

17. The assembly of claim 1, wherein each of the plurality of wells is a microfluidic device and comprises 50 to 400 micro-channels such that the CMAP assembly has a high throughput.

18. The assembly of claim 1, wherein the plurality of micro-channels are sized for tumor cells to squeeze through the micro-channels to provide three-dimensional (3D) migration.

19. The assembly of claim 1, wherein each of the plurality of troughs includes opposing side walls and a bottom wall extending between the opposing side walls.

20. The assembly of claim 1, wherein each of the plurality of micro-channels includes a square cross-section or a rectangular cross-section.

21. A method of fabricating a cell migration assay plates (CMAP) assembly, the method comprising:

forming a top plate having a plurality of wells, each of the plurality of wells defined at least in part by a first reservoir, a second reservoir, and a divisional wall extending between the first reservoir and the second reservoir;

forming a bottom plate having a plurality of troughs; and

forming a plurality of micro-channels by securing the top plate to the bottom plate, each of the plurality of micro-channels defined by a portion of the divisional wall of each of the plurality of wells and middle portions of the plurality of troughs, the plurality of micro-channels enabling communication between the first reservoir and the second reservoir when the bottom plate is secured to the top plate.

22. The method of claim 21, further including:

aligning the top plate and the bottom plate so the divisional wall abuts the plurality of troughs.

23. The method of claim 21,

wherein, the top plate is formed via an injection molding process; and the bottom plate is formed via a hot embossing process.

24. The method of claim 21, wherein the bottom plate is formed using an optically translucent or transparent biocompatible cyclic olefin polymer (COP) to enable viewing of cell migration in the micro-channels.

25. The method of claim 21, wherein the top plate is formed using a black cyclic olefin polymer (COP).