WO2011035185A2

WO2011035185A2 - A microfluidic device and uses thereof

Info

Publication number: WO2011035185A2
Application number: PCT/US2010/049372
Authority: WO
Inventors: Ali Khademhosseini; Masoud Khabiry; Bong Geun Chung; Matthew Hancock; Yanan Du
Original assignee: Brigham and Womens Hospital Inc
Current assignee: Brigham and Womens Hospital Inc
Priority date: 2009-09-17
Filing date: 2010-09-17
Publication date: 2011-03-24
Anticipated expiration: 2012-03-17
Also published as: WO2011035185A3

Abstract

Microfluidic devices and methods for using microfluidic devices for immobilizing cells, e.g., mammalian cells, suspended in a flowing fluid include selecting the geometry and dimensions of one or more multi-surface cavities disposed therein, and/or the flow direction of a cell-containing fluid therein. The cavity can comprise an upper and lower surface, and cells can be captured on either surfaces located at two different depths within a cavity. The invention includes methods for generating co-cultures of different cell populations inside a multi-surface cavity disposed within microfluidic devices as disclosed herein.

Description

A MICROFLUIDIC DEVICE AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This International application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 61/243,293, filed September 17, 2009, the contents of each of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates generally to microfluidic devices and methods for immobilizing cells from a flow, e.g., by accumulating cells in microstructures therein. In some embodiments, the invention relates to microfluidic devices comprising cavities with at least two surfaces and methods for co-culturing cells.

BACKGROUND OF THE INVENTION

[0003] The in vitro manipulation, study and processing of living cells continues to be of importance both for theoretical evaluation as well as for the in vitro assaying of compounds for biological activity in such cells. However, conventional biological assay systems such as tissue culture dishes and cell perfusion chambers are typically operated with large volumes of cells and/or reagents, e.g., from 1 ml to 100 ml of reagents or more. In circumstances where the source of cells is limited, e.g., primary cells isolated from tissues, it is very difficult to use conventional assay systems with such precious cells. Further, statistical variations within a large population of cells can limit the ability to resolve the effect of a compound, e.g., a drug, on biological activity of cells.

[0004] Recently small disposable devices have been developed for handling biological samples and for conducting in vitro experiments on a controlled basis. For example, microfluidic devices are developed for high-throughput screening and biochemical synthesis. They enable the controlled manipulation of small amounts of fluid, and minimize consumption of costly reagents. Thus, microfluidic devices are suitable for cellular applications due to their small size and high-throughput capability. Moreover, the cellular microenvironment within microfluidic devices can be controlled by regulating different parameters such as flow rate and shear stress.

[0005] Previous microfabrication-based techniques for cell immobilization within microchannels include photocrosslinkable hydrogel patterning [13,14], cup-shaped cell isolation arrays [15], and microwell structures [9]. An alternative approach to capture cells within fluidic system is through the use of single-grooved substrates that create regions of low shear stress. Microdevices with shear-protective single-grooved substrates enable cell capture, and cell exposure to soluble factors, features necessary for cell-based applications such as drug discovery [9,10] and microbioreactors [11,12]. However, it is often desirable to array cells at discrete and separable locations, so that automated assays can be performed easily and the results can be observed at a single cell level. The cell of interest can then also be further isolated and clonally expanded.

[0006] In prior-art devices having such single-grooved substrates, cell positions and microcirculations could be controlled by varying the flow rate and the groove aspect ratio [16]. However, in single-grooved substrates, cells are captured only in the bottom surface of the grooves. It is impossible to accumulate cells at various depths within a cavity. In addition, the width of the bottom surface of single-grooved substrates is a determinant of a cell position within a microsurface. Hence, the single-grooved configuration does not allow flexible control of cell capture or co-cultures of more than two cell types.

[0007] Co-cultures are useful for studying homotypic and heterotypic cell-cell interactions and enhancing the function of cell types that are hard to maintain in vitro (such as hepatocytes) through introduction of support cells that provide the signals to maintain these cells in culture. Previous approaches to fabricate multiphenotype arrays involved a number of techniques such as patterned co-cultures and capturing cells within photocrosslinking or natural polymers. In patterned co-cultures, two cell types are positioned relative to each other, either by using selective adhesion of one cell type relative to the other to a patterned substrate or by using the reversible adhesive properties of the substrate to position a cell type relative to the other cell type. However, these techniques usually require patterning substrate surfaces with cell adhesion molecules, and most patterned co-cultures to date only employ two cell types patterned relative to each other.

[0008] Although immobilization of cells within photocrosslinkable hydrogels using an injection molding technique has been used to pattern multiple cell types on a two dimensional substrate. Some potential challenges include the use of toxic photoinitiators and radiation to immobilize the cells inside the channels and the need for expensive photolithographic patterning equipment. Further, by photocrosslinking the cells in a hydrogel, it is harder to retrieve the cells for subsequent analysis. Therefore, there is a strong need for an improved design of a simple yet adaptable microfluidic device that can be used for high throughput immobilization of one or more cell type within the same microwells. SUMMARY OF THE INVENTION

[0009] The present invention is generally directed to microfluidic devices and methods for capturing living cells, e.g., mammalian cells, on one or more surfaces within at least one cavity of a microfluidic device, for example, by modulating the geometry of a multi-surface cavity disposed therein, and/or the flow direction of a cell-containing fluid therein. In one embodiment, a microfluidic device can immobilize cells by capturing and immobilizing cells on one or more surfaces located at two or more different depths within a cavity. In some

embodiments, the invention provides methods for generating co-cultures of different cell populations inside a multi-surface cavity disposed therein.

[0010] In accordance with the invention, the position of cell capture can be controlled by adjusting the ratios of depths of two or more surfaces of a cavity in a microfluidic device. For example, cells tend to accumulate upstream on deep and narrow surfaces, and tend to

accumulate downstream on shallow and wide surfaces. Further, deeper surfaces within a cavity tend to increase cell retention. Accordingly, devices according to embodiments of the invention can include a microfluidic device with multi-surface microstructures that generate shear- protected regions, enabling cell capture at different depths within microstructures.

[0011] One aspect of the invention relates to a microfluidic device for cell capture on one or more surfaces at different depths within a cavity. In some embodiments, a microfluidic device comprises a base substrate having a top surface and at least one cavity in the top surface. In various embodiments, a cavity comprises at least a first surface and a second surface, wherein the first surface is located at a predefined distance hi below the top surface, and the second surface is located at a predefined distance h2 below the first surface. In a further embodiment, a microfluidic device comprises a top substrate mounted on the top surface of the base substrate. In such embodiments, the top substrate can include one or more channels extending over the base substrate, and each channel can have a predefined height ho or a height profile (with a predefined height ho relative to the cavity), wherein each channel communicates with one or more cavities on the base substrate.

[0012] Another aspect of the invention relates to the design and fabrication of a microfluidic device as disclosed herein. In various embodiments of a cavity therein, the first and the second surface can have irregular shape or a regular shape. Non-limiting examples of a regular shape include a circle, a square, a rectangle, a triangle, a trapezoid, a polygon and the like. In some embodiments, the first and the second surface can be concentric. In alternate embodiments, the first and/or the second surface can be asymmetric. In further embodiments, the dimensions hi and h2 can be greater than a cell diameter, e.g., at least about 20um. In one embodiment, the ratio of h2 to hi can be greater than 1, e.g., any continuum values greater than 1. In an alternative embodiment, the ratio of h2 to hi can be smaller than 1, e.g., any continuum values smaller than 1. In another embodiment, the ratio of h2 to hi can be equal to about 1. In various embodiments, the ratio of h2 to hi can be an integer and/or a fraction.

[0013] A further aspect of the invention includes methods for capturing and

immobilizing cells on one or more surfaces located at various depths within at least one cavity in a microfluidic device as disclosed herein. The method includes flowing a cell-containing fluid on top of a surface having at least one cavity therein. In one embodiment, a cavity comprises one or more surfaces located at two or more two different depths within a cavity. In some

embodiments, at least one cell can be captured on the first surface of a cavity. In alternative embodiments, at least one cell can be captured on the second surface of a cavity. In one embodiment, when the ratio of h2 to hi can be greater than 1, at least one cell is captured on upstream portion of a cavity. In another embodiment, when the ratio of h2 to hi is smaller than 1, at least one cell is captured on downstream portion of a cavity. In various embodiments, the ratio of h2 to hi can be an integer and/or a fraction. In further embodiments, flow rate of a cell- containing fluid can be modulated to adjust cell retention, including the duration of time a cell can be retained on a surface when subjected to a flow.

[0014] Yet another aspect of the invention is directed to methods for co-culturing cells in a cavity with multiple surfaces at various depths therein in a microfluidic device as disclosed herein. The method comprises adjusting the flow direction of a cell-containing fluid and/or aspect ratios of a cavity. In such embodiments, to create a co-culture, a second cell-containing fluid with a different cell type can be introduced into a microfluidic device of the invention with a flow direction different from that of the first cell-containing fluid.

DESCRIPTION OF THE DRAWINGS

[0015] Figures 1A and IB show schematics of a microfluidic device of the invention. Figure 1A illustrates a top substrate with a channel (top drawing) and a base substrate having a top surface with cavities (middle drawing) including a close-up of cavities with two surfaces at different depths therein (bottom drawing). Figure IB shows one embodiment of a

microfabricated device as disclosed herein with cells captured in cavities (top drawing), as well as a close-up of cells inside the cavities (bottom drawing). Color versions of drawings are available in Khabiry et al., X Small. 1 (2009). [0016] Figures 2A to 2C show the top view and side view of a cavity having two surfaces at different depths therein. Figure 2A is a SEM image of four cavities, each of which has two surfaces at different depths therein. Figure 2B is a SEM image of a cross-section of a cavity having two surfaces at different depths therein. Figure 2C shows a schematic design of a cavity having two surfaces at different depths therein, with inlet channel width τνθ and height hO and surface widths (1, (2, lateral lengths τνΐ, u>2, and heights hi, h2. Examples of corresponding dimensions of fabricated cavities are listed in Table 1 in Example 1.

[0017] Figures 3A to 3C show numerical simulation of flow in a cavity having two surfaces at different depths therein. Figure 3A shows a computational domain including geometry and triangular mesh discretization. Figure 3B shows streamlines and velocity contours for the five characteristic flow patterns in a cavity having two surfaces at different depths therein. Configuration (i) has h2lhl = 0.1, hOlhl = 8/15, (hl+h2)/il = 2/3 and (2/(1 = 0.5.

Configuration (ii) has h2lhl = 0.1, hOlhl = 0.5, (hl+h2)/(l = 2/3 and (2/(1 = 0.5. Configurations (iii- v) correspond to the fabricated geometries 1-3, respectively, listed in Tables 2 and 3 (See Example 2). Arrows indicate flow direction. Figure 3C shows ratio of shear stress along the bottom surface, _ot, to that of the inlet channel, To, for configurations (iii-v), h2lhl=H (curve 320), l(curve 330), 4 (curve 350). Corresponding profiles for single-groove limits h2/hl—>0 (curve 310) and hllh2→ 0 (curve 350) are shown. The x-axis is the horizontal coordinate parallel to the flow and scaled with respect to the upper surface width.

[0018] Figures 4A to 4E show cell capture in cavities having two surfaces at different depths therein. Figures 4A to 4C show representative top views of cell positions on the lower and upper surfaces for different geometries (β=200μπι). Figures 4D and 4E show cell counts on upstream and downstream portions of the upper and lower surfaces, for geometries for narrower (β=150μπι) and wider surfaces (β=200μπι), respectively. Schematics of experimental setup are shown on the left of Figures 4D and 4E, while those of the flow and cell capture patterns are shown below thereof.

[0019] Figures 5A to 5D show experimental analysis of cell retention in cavities having two surfaces at different depths therein. Figures 5A and 5B show cell retention for various inlet flow velocities U in narrow (/ί=150μπι, β=75μπι) and wide (#=200μπι, β=100μπι) surfaces, respectively. Values are normalized by the number of cells on surfaces at U=0.052cm/s listed over appropriate bars. Figures 5C to 5D show representative cell retention measurements as a function of U on lower surfaces and upper surfaces, respectively. Fitted lines (dashed or solid lines) demonstrate the linear dependence on U for each stage of the cell-removal process. The asterisk (*) indicates maximum retention for previously designed devices comprising cavities with one surface [16].

[0020] Figures 6A to 6E show phase diagrams of the characteristic flow patterns in cavities having two surfaces at different depths therein, with experimental results of cell capture patterns in cavities with two- and single- [16] surface geometries (parameters listed in Table 2 in Example 2). Figure 6A is a phase diagram of the characteristic flow patterns for (hl+fi2)l[l =1/2 with data for (unfilled triangle) upstream, (filled triangle) downstream capture of cells, and (filled circle) single surfaces [16] with downstream capture of cells. Figure 6B is a phase diagram of the characteristic flow patterns for ( LZ+ L2)/fl=2/3 with data for (unfilled square) upstream and (filled square) downstream capture of cells. Figure 6C shows a phase diagram for single surfaces ( L2=0) with data [16] for (unfilled circle) upstream and (filled circle) downstream capture of cells. Flow patterns are illustrated by schematics (insets) and denoted by roman numerals i-v corresponding to configurations discussed in Figure 3B. The asterisk (*) denotes single- surface limit ( L2=0) of phase boundary. Figure 6D shows dependence of phase boundaries on surface width ratio [2/ [1=0.4 (dashed line,— ), 0.5 (solid line, ), 0.6 (broken lines with a dot between gaps, - · -). Figure 6E shows dependence of phase boundaries on total groove depth

( LZ+ L2)/fl=l/2 (solid line, ), 2/3 (dashed line,— ), 5/6 (broken lines with a dot between gaps, - · -), 1 (long dash). Reynolds number Re in Figures 6A to 6C is 0 (solid line, ), 0.1

(dashed line,— ), 1.0 (broken lines with a dot between gaps, - · -) and in Figures 6D to 6E, Re=0.

[0021] Figures 7A to 7C shows the effect of alignment of the bottom surface within a cavity on flow pattern. Figure 7A shows streamlines for up stream- shifted, centered, and downstream-shifted bottom surfaces for fabricated surface geometry 4 (Table 2 in Example 2, (Hl+H2)/[1 =2/3, β/β=0.5, H0/fl=S/15) and Re=0.1. Shifts were quantified by distance between surface centers as a fraction of upper surface width. Ends of computational domain (channel ends) are not displayed. Figures 7B to 7C show the effect of surface alignment on flow pattern phase boundaries at Re=0 for (hl+fi2)l[l =2/3 and 1/2, respectively. Phase boundaries for upstream/downstream shifts of same magnitude coincide and flow patterns are mirror images about surface center. Experimental data indicate observed cell-capture pattern on narrow

((Hl+H2)/[1 =2/3) and wide ((Hl+H2)/[1 =1/2) cavities with two surfaces (Table 2 in Example 2).

DETAILED DESCRIPTION OF THE INVENTION [0022] In some embodiments, the present invention is based upon the discovery that the location of cell capture can be controlled by selecting the geometry and the dimensions of two or more surfaces within a cavity in a microfluidic device. In accordance with the invention, cells can be captured upstream on deep and narrow surfaces, and can be captured downstream on shallow and wide surfaces. In addition, deeper surfaces increase cell retention.

[0023] Accordingly, in some embodiments, the present invention provides microfluidic devices with cavities comprising multiple surfaces at various heights therein (shear-protected regions) for cell capture. In some embodiments, the present invention relates in part to the design and fabrication of a microfluidic device as disclosed herein. Another aspect of the present invention provides methods for cell capture in a cavity having two or more surfaces at two or more depths therein in a microfluidic device as disclosed herein. Yet another aspect of the invention is directed to methods for co-culturing cells in such cavities in a microfluidic device as disclosed herein. Still another aspect of the invention relates to the use of a microfluidic device as disclosed herein for making co-cultures and for cell-based biosensing and drug screening.

[0024] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Definitions

[0025] The term "substrate" as used herein includes a support material on which cavities or channels are disposed therein. In some embodiments, the substrate can be made of any material such as glass, co-polymer or polymer. Exemplary polymers include, but not limited to, urethanes, rubber, molded plastic, polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, and the like. In one embodiment of the invention, a substrate can be made of polydimethylsiloxane (PDMS). Such substrate materials have advantages of their ease of manufacture, low cost and disposability, as well as their general inertness to most extreme reaction conditions. These materials can include treated surfaces, such as, derivatized or coated surfaces, to enhance their utility in the microfluidic system, e.g., provide enhanced fluid direction, e.g., as described in U.S. Pat. No. 6,238,538, and which is incorporated herein by reference in its entirety for all purposes. For example, in some embodiments of the invention, the inner walls and surfaces of a cavity can be coated with one or more cell adhesion molecules or agents, e.g., fibronectin, collagen, laminin, gelatin, or RGD to improve cell adhesion to a substrate, using the methods known in the art. In further embodiments, inner walls of such cavities can be treated with a material to modify their hydrophilicity, protein affinity, cell affinity, or any combination thereof. Exemplary materials, include but not limited to, poly(3- trimethoxysilyl)-propylmethacrylate-r-poly(ethylene glycol) methyl ether (TMSMA-r-PEGMA), organosilanes that form self-assmebled monolayers, or ethanol.

[0026] The term "surface" as used herein in reference to a cavity refers to a plane or curved surface within the cavity. In one embodiment, the surfaces within the cavity can be parallel to the top surface and/or the bottom surface of a base substrate. In some embodiments, the surfaces within the cavity can be non-parallel to a top surface and/or a bottom surface of a base substrate. It should be understood that in the description and figures illustrated herein, the cavities disposed in a microfluidic device are generally shown as having planar surfaces;

however, structures fabricated from flexible and/or elastomeric materials that are capable of being bent, twisted, or distorted from the illustrated planar configurations. For such

embodiments, the "surfaces" within a cavity can comprise curved surfaces, and any discussion herein with regard to "surfaces" of the cavities should be understood to encompass such curved surfaces as well as the planar surfaces illustrated.

[0027] The phrase "a multi-surface cavity" as used herein refers to a cavity disposed in a microfluidic device having a top surface, the cavity comprising at least a first surface and a second surface, wherein the first surface is located at a predefined distance hi below the top surface, and the second surface is located at a predefined h2 below the first surface. For example, Figures 2A to 2C illustrate a cavity having an upper and a lower surface. Similarly, the term "single surface" as described herein refers to a cavity having one surface with h2 = 0. The terms "upper surface" and "lower surface" when used in reference to a cavity refer to the first face surface located at a predefined distance hi below the top surface, and the second surface located at a predefined h2 below the first surface, respectively. The cavity can include more than two surfaces.

[0028] The term "width" when used in reference to a cavity refers to the width of a surface within a cavity measured along the flow direction, and is denoted by ί followed by a number indicative of a numbered surface, e.g., [1 for the first surface. In some embodiments, for a triangular cavity, the width of a surface within a cavity can change along the cross-section of the flow. Similarly, the surface width of a circular or an oval cavity refers to the width of the surface covered by the fluid along the flow direction. Figure 2C illustrates the surface widths (il and [2) of a cavity having an upper and a lower surface.

[0029] The term "perimeter" when used in reference to a cavity refers to the border of a surface located below a top surface of a base substrate when viewed from the top. Figure 2C illustrates that the perimeter of the first and second surface within a cavity has a rectangular shape. Without wishing to be bound by theory, the perimeter of surfaces within a multi-surface cavity can have any shape. Examples of shapes include, but not limited to, a circle, an ellipse, a triangle, a square, a rectangle, a parallelogram, a trapezoid, a rhombus, a polygon and the like. In some embodiments, the perimeter of the surfaces can have irregular shape.

[0030] The terms "immobilize", "capture", "capture" or "position" as used

interchangeably herein, refer to cells in a suspension or in a flow adhering to a substrate and not washed away by a flow. For example, Figures 4A to 4C demonstrate immobilization of cells from a flow on one side of the cavities as the cells are captured and accumulated upstream or downstream of multi-surface cavities. By adjusting the ratio of h2 to hi, cells can be positioned upstream or downstream of multi-surface cavities accordingly.

[0031] The phrase "shear protection" as used herein refers to protection from shear stress generated by a fluid flow. According to some embodiments of the invention, a multi-surface cavity offers cells a better shear protection from the fluid flow than those in a cavity with one surface only. Thus, cell retention is higher in a multi-surface cavity than in a cavity with one surface only.

[0032] The term "cell-containing fluid" used herein is defined as any liquid carrier of cells, including all types of cell culture media with or without serum, and also includes blood, plasma, urine, as well as buffered isotonic solutions capable of supporting cell suspensions. A cell-containing solution can also be an environmental sample containing microorganisms from bodies of water such as lakes, ponds, streams or oceans, etc. The term "cell culture medium" or "cell culture media" (also referred to herein as a "culture medium" or "medium") as used herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium can contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art. In some embodiments, cell culture medium can include serum or not include serum. [0033] The term "cell" includes both eukaryotic and prokaryotic cells, including bacteria, yeast, mammalian cells etc. In some embodiments, the cells are eukaryotic cells. In other embodiments, the cells are mammalian cells. Other cells include cardiac muscle cells, fibroblasts or any cells which can be suspended in a single cell suspension. A cell can come from a cell line or be derived from a primary tissue culture.

[0034] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0035] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The present invention is further explained in detail by the following, including the Examples, but the scope of the invention should not be limited thereto.

Microfluidic device and fabrication thereof

[0036] Microfluidic devices can be used for high-throughput screening and biochemical synthesis [1-4]. These devices manipulate fluid flows, minimize costly reagent consumption, and enable high-throughput experimentation in a controlled manner [1,3,5,6]. Moreover, the cellular microenvironment within microfluidic devices can be controlled by regulating different soluble factors such as the flow rate and shear stress [7,8].

[0037] One aspect of the invention provides microfluidic devices that provide (1) controlled cell capture and potential cell immobilization on one or more surfaces of at least one cavity therein, (2) better shear protection of cells from being washed away by a fluid flow, and (3) capability of co-culturing different cell types. In one embodiment, e.g., in Figures IB and 2C, a microfluidic device comprises a base substrate 10 having a top surface 32 and at least one cavity 30 in the top surface. In such embodiments, the cavity 30 comprises a first surface 34 and at least a second surface 36, wherein the first surface 34 is located at a predefined distance hi below the top surface 32; and the second surface 36 is located at a predefined distance h2 below the first surface 34. In one embodiment, a base substrate 10 with a top surface 32 comprises a plurality of cavities. [0038] In accordance with the invention, the ratio of h2 to hi is important for

determining location of cell capture. When dimensions of a cavity such as hi and h2 are greater than a cell diameter (or a cell diameter is small relative to cavity dimensions, e.g., hi and h2), the cell capture pattern can be predicted solely based on the microcirculation pattern determined by computational analysis as shown in Example 2. For example, the larger depth ratios h2lhl are associated with microcirculation in the bottom cavity, resulting in cells accumulating on the upstream surfaces. Smaller depth ratios h2lhl correspond to little or no microcirculation and hence cells accumulate on the downstream surfaces. Furthermore, cells are more dispersed in the deeper bottom surfaces as a result of the higher shear protection and weaker flows.

[0039] Accordingly, in some embodiments, hi and h2 are each greater than a cell diameter. In some embodiments, hi can be at least about ΙΟμπι, at least about 20μπι, at least about 30μπι, at least about 40μπι, at least about 50μπι, at least about 60μπι, at least about 70μπι, or at least about 80μπι. In various embodiments, h2 can at least about at least about ΙΟμπι, at least about 20μπι, at least about 30μπι, at least about 40μπι, at least about 50μπι, at least about 60μπι, at least about 70μπι, or at least about 80μπι. In some embodiments, hi or h2 ranges from about 20um to about 80um.

[0040] Depending on a desired location of cell capture (e.g., upstream or downstream of a cavity), the ratio of h2 to hi can be selected accordingly. In one embodiment, if cell capture on the upstream portion of a cavity is desirable, the ratio of h2 to hi can be an at least about 1 or any continuum value greater than 1. In one embodiment, the value can be an integer, e.g., about 1, about 2, about 3, about 4, about 5, or about 6. In another embodiment, the value can include an integer plus a fraction, e.g., about 1.5, about 2.4, about 3.6, about 4.3, about 5.8, about 6.2. In alternative embodiments, if cell capture on the downstream portion of a cavity is desirable, the ratio of h2 to hi can be any continuum value less than 1 and the value can be any fraction. In one embodiment, the ratio of h2 to hi can be about 0.1, about 0.2, about 0.25, about 0.3, about 0.4 or about 0.5. Depending on the flow condition (e.g. Re) and other aspect ratios (h0/[l or (hl+h2)/[l)) as demonstrated in Figures 6A to 6E and Example 4, the ratio of h2 to hi can be smaller than 1 for upstream capture of cells. Similarly, the ratio of h2 to hi can be greater than 1 for

downstream capture of cells.

[0041] In various embodiments, a microfluidic device of the invention further comprises a top substrate 20 mounted on the top surface of the base substrate 10, wherein the top substrate defines at least one channel 22 extending from an inlet 24 to an outlet 26 over the base substrate 10. In such embodiments, the at least one channel 22, with a predefined height ho, is in communication with the cavity 30 on the base substrate 10 and the at least one channel 22 has a predefined height hO or a height profile (with a predefined height hO relative to the cavity). In some embodiments, hO can have a height of about 20 μπι to about 1 mm. In some embodiments, hO can be at least about 20μπι, at least about 30μπι, at least about 40μπι, at least about 50μπι, at least about 60μπι, at least about 70μπι, at least about 80μπι, about 90μπι, about ΙΟΟμπι or about 500μπι. In one embodiment, hO can be about 80um.

[0042] In further embodiments of a top substrate, the channel extending over the base substrate can have a width of 1 μπι to 1 mm, for example, between 1 and 10 um, between 10 and 100 um, between 100 and 500 um, or between 500 um and 1 mm. The optimal channel size can depend, e.g., on the size of the cavities and the experimental condition, for example, the size of the cells.

[0043] In one embodiment, a top substrate includes one channel extending across the base substrate and providing fluid communication with one or more cavities in the base substrate, as illustrated in Figure 1. In some embodiments, one or more additional channels in the top substrate can be formed in parallel to the first channel. In other embodiments, the additional channel in the top substrate can be formed orthogonal to the first channel. In further embodiments, the top substrate can comprise an array of channels, wherein the channels can be positioned in parallel and/or orthogonal to one another.

[0044] In various embodiments, a microfluidic device as disclosed herein comprises a cavity having at least a first surface and a second surface, wherein the first surface has a predefined width il and the second surface has a predefined width [2, wherein il is greater than

[2. In some embodiments, the ratio of il to [2 can be greater than 1. In some embodiments, il or

[2 can be further increased to increase the number of cells captured on the first and/or second surface. In one embodiment, the ratio of il to [2 can be about 2, about 2.5 or about 3.

[0045] In further embodiments, il and [2 can be determined as a function of hO, hi, h2, a desired number of cells on each of the first and the second surfaces, corner eddies and shear stress profile in the cavity, and/or properties of fluid. The desired number of cells on each of the first and the second surfaces can be determined as a function of binding affinity of cells to substrates, cell retention time, flow rate of a fluid passing through a channel in the top substrate and surface area for cell capture. For example, decreasing [2 or increasing h2 can enhance shear protection and thus cell capture. Accordingly, if binding affinity of a cell to a substrate is weak, in accordance with the invention, cell capture can be improved without coating the cavity with any cell adhesion molecules by decreasing [2 , increasing h2 or a combination thereof . [0046] In yet further embodiments, ho can be determined as a function of il and properties of fluid, e.g., flow rate of a fluid passing through the channel in the top substrate, the density and viscosity of the fluid. In some embodiments, the ratio of ho to il can be less than 1. In other embodiments, the ratio of ho to il can be in the range of about 0.2 to about 0.7. In further embodiments, if the flow rate of the fluid is increased, ho can be increased to maintain the same shear stress on the inlet channel wall as with the lower flow rate. Similarly, if the viscosity or density of the fluid is increased, ho can be also increased to keep the same shear stress on the inlet channel wall as with the lower fluid viscosity or density.

[0047] In some embodiments of a microfluidic device as disclosed herein, a cavity comprises at least a first surface and a second surface, wherein the perimeter of the first surface and the second surface can have any shape. For example, the perimeter of the first and/or the second surfaces can have a regular shape, for example, a square, a triangle, a polygon and the like. The term "polygon" as disclosed herein refers to a shape bounded by three or more straight lines that meet in pairs at the vertices but do not intersect other than at these vertices. In another embodiment, the perimeter of the first and the second surfaces can have irregular shape. The term "irregular shape" as disclosed herein refers to any shape that is not a regular shape. In some embodiments, an irregular shape can be symmetric or asymmetric with respect to an axis, such as the direction of flow transverse to the direction of flow.

[0048] In various embodiments, a cavity in a base substrate of a microfluidic device of the invention comprises at least a first surface and a second surface, wherein the first surface has a predefined lateral length wl, and the second surface has a predefined lateral length w2. In some embodiments, wl and w2 can be independently between about 20 μπι and about 5mm, for example, between about 30um and about 4.5mm, between about 40um and about 4mm, between about 50um and about 3mm, or between about 60um and about 2 mm. The optimal size of a cavity can be adjusted depending on the cell type to be employed in the method, for example, the cell size or degree of cell adhesion to the cavities, or the experimental condition that will take place within the cavities. In some embodiments, when a cavity has a circular or oval shape, wl can be equal to about il and/or w2 can be equal to about [2, depending on the flow direction. In other embodiments, a square-shaped cavity can have wl equal to about il and u>2 equal to about [2, depending on the flow direction. In further embodiments, a cavity of a triangular shape can have wl and w2 in reference to heights of triangles at different levels within a cavity, respectively. [0049] When fluid flows past a sharp corner in a multi-surface cavity, cells can be effectively shear-protected and captured on a surface if the surfaces formed inside the cavity afford sufficient shear protection. In one embodiment, the perimeter of the first surface and the second surface has a rectangular shape, and the cross-section of the cavity is illustrated in Figure 2B. Without wishing to be bound by theory, a myriad of geometries can be employed to create various cell capture patterns, such as multi-surface cavities of rectangular, triangular, trapezoidal, or other cross sections (see Reference [16] and references therein). Computational flow simulations as disclosed in Example 2 can be used to predict the patterns of cell capture and the degree of shear protection. Accordingly, an optimal choice of cavity geometry and aspect ratios can be determined by computational analysis, based on a number of variables such as concomitant cell-capture pattern, fabrication viability, mass-transport properties, and the area afforded for cell adhesion. Such features are important for the design of biosensors in order to deliver nutrients and diagnostic chemicals to cells captured on one or more surfaces within a cavity therein. One skilled in the art will recognize that it may be desirable to use lithographic and other techniques to form cavities of various shapes.

[0050] In some embodiments of a microfluidic device as disclosed herein, a cavity comprises at least a first surface and a second surface that can be concentric, i.e., the centers (or the centers of mass) of a first and a second surfaces are aligned. For example, Figures 2A to 2C illustrates a cavity of two concentric rectangular surfaces. In accordance with the invention, in a cavity with a symmetric geometry, e.g., in Figures 2A to 2C and 3 A to 3C, the flow streamlines and velocity profiles of the flow can be symmetric or nearly symmetric. In other embodiments, misalignment between the centers of the first and second surfaces can break the symmetry of the cavity geometry and the near symmetry of the flow. An exemplary example of misaligned surfaces or an asymmetric geometry is shown in Figure 7A. However, in accordance to the invention, a numerical simulation of a cavity having an upper and lower surfaces demonstrates that misalignment of up to about 1/8 of the width [1 of the first surface does not alter the characteristic flow patterns for designs that are relatively wide (( LZ+ L2)/fl=l/2) or have a deep second surface H2lHl>l). In one embodiment, the first and second surfaces can be misaligned by up to about 1/2 of the width [1 of the first surface, up to about 1/4 of the width [1 of the first surface or up to about 1/8 of the width [1 of the first surface.

[0051] In a further embodiment, a second surface can be shifted completely to one side, an edge and a portion of the upper surface disappear along with the associated eddies. The effects of misalignment between surfaces of a cavity on the flow pattern can be precisely assessed with numerical simulation as described in Example 2, and the position of cell capture can be predicted. Accordingly, some embodiments of the invention provide a theoretical framework for the design of a microfluidic device comprising cavities with at least two surfaces to achieve desirable cell capture. As the flow pattern and shear stresses influence the pattern of cell capture, an optimal configuration of cavities disposed within microfludic device can be determined by computation analysis, e.g. numerical simulation in Example 2.

[0052] Another aspect of the invention relates to methods for fabrication of a

microfluidic device as disclosed herein. In some embodiments, the method for making a microfluidic device of the invention comprises: (1) providing a base substrate having a top surface; (2) constructing at least one cavity in the top surface, the cavity comprising at least a first surface and a second surface; (3) forming the first surface at a predefined distance hi below the top surface; (4) forming the second surface at a predefined distance h2 below the first surface; and (5) mounting a top substrate on the top surface of the base substrate, the top substrate including at least one channel in communication with the cavity on the base substrate, the channel having a predefined height ho.

[0053] In various embodiments, a base substrate can have at least 1 cavity, at least 2 cavities, at least 3 cavities, at least 4 cavities, at least 5 cavities, at least 10 cavities, at least 20 cavities, at least 40 cavities, at least 80 cavities, at least 100 cavities, at least 200 cavities or at least 300 cavities.

[0054] In some embodiments, the cavity includes at least a first surface formed at hi below the top surface of the base substrate and a second surface formed at h2 below the first surface. In one embodiment, the cavity can have two surfaces at two different depths thereof. In accordance with the invention, cell retention is less the on upper cavity surface than on the lower cavity surface. Accordingly, in some embodiments, a cavity can have more than 2 surfaces to improve cell capture on at least one surface of the cavity. For example, a cavity can have a first surface located at hi below the top surface of the base substrate, a second surface located at h2 below the first surface, and a third surface at h3 below the second surface, and so on. In additional embodiments, additional surfaces located at a predefined distance below a respective upper surface can be formed in a cavity. Stated in another way, a cavity can have a bottom surface and a sidewalk In some embodiments, at least one portion of the sidewall comprises more than 1 step, more than 2 steps, more than 3 steps, more than 4 steps, more than 5 steps, more than 6 steps, more than 10 steps, or more than 20 steps. In one embodiment, a cavity can have one step. To determine the number of surfaces within a cavity required for a desired flow pattern and cell capture locations, computational simulation of a fluid flow in a designed geometry can be performed as disclosed, e.g., in Example 2. [0055] In further embodiments, a base substrate can comprise one or more cavities. In additional embodiments, a base substrate can comprise identical cavities or different cavities. In such embodiments, different cavities can have various cavity shapes, different number of steps or surfaces, and/or a variety of cavity dimensions. In further embodiments, the cavities can be randomly arranged in a base substrate. In another embodiment, the cavities can be arranged along the channel in the top substrate. In other embodiments, the cavities can be orderly arranged in a base substrate, e.g., in a matrix (N x N) configuration. For example, Figures 1A and IB show cavities arranged in a 1x7 matrix configuration. In some embodiments, each cavity in a base substrate can communicate with at least one channel in a top substrate. For example, for a rectangular- shaped cavity, two orthogonal channels can be desirable when cell capture on all four sides of the cavity is preferred. In other circumstances, multiple channels, having a channel width τνθ smaller than τνΐ and/or u>2, in communication with the same cavity can allow simultaneous flow of multiple fluids. In such embodiments, the fluid flowing through each channel can be cell-containing fluid comprising one or more cell populations. Accordingly, different cell populations can be simultaneously captured on one or more surfaces of the cavity. In another embodiment, after cell captured on surfaces of cavities disposed within a microfluidic device as disclosed herein, the fluid flowing through each channel, having a channel width τνθ smaller than τνΐ and/or u>2, in communication with the same cavity can be a cell-free fluid. In such embodiments, the cell-free fluid flowing through each channel can be cell culture medium containing a different agent, e.g., a growth factor or a drug or cell adhesion molecules. Hence, one or more agents can be separately delivered to the cells within the cavity and mixed inside the cavity. This can eliminate tedious liquid mixing or handling during experimentation.

[0056] In accordance with embodiments of the invention, a top substrate can comprise at least one channel, at least 2 channels, at least 3 channels, at least 4 channels, at least 5 channels, at least 10 channels, at least 20 channels, or at least 30 channels. In some embodiments, the number of channels in a top substrate and the number of cavities in a base substrate can be coordinated. Channels can be oriented in multiple configurations. For example, channels can be arranged in an ordered array, in an orthogonal arrangement, in a random configuration, or in a combination thereof.

[0057] A top substrate and a base substrate of the invention can be fabricated by photolithography and standard soft lithography techniques. See Love, et al., MRS BULLETIN, pp. 523-527 (July 2001) "Fabrication of Three-Dimensional Microfluidic Systems by Soft Lithography", Delamarche et al,: JOURNAL OF AMERICAN CHEMICAL SOCIETY, Vol. 120, pp. 500-508 (1998), Delamarche et al,: SCIENCE, Vol. 276, pp. 779-781 (May 1997), Quake et al., SCIENCE, Vol. 290, pp. 1536-1540 (Nov. 24, 2000), U.S. Pat. 6,090,251, all of which are hereby incorporated by reference for purposes of techniques for soft lithography and microfabrication. Alternatively, substrates can be readily manufactured from fabricated masters, using well known molding techniques, such as injection molding, embossing or stamping, or by polymerizing the polymeric precursor material within a mold.

[0058] For photolithography, a master mold can be made to produce a negative of the pattern desired for a top substrate (e.g., with a channel disposed therein) or for a base substrate (e.g., with a plurality of cavities disposed therein). For example, to create one channel in a top substrate, the master mold can be patterned to form one raised channel to produce a hollow channel that communicates with at least one cavity in the base substrate. Accordingly, a master mold can be patterned to form one or an array of raised channels to produce a desired number of hollow channels in a top substrate of a microfluidic device as disclosed herein. Similarly, a master mold can be created to form one or a plurality of cavities in a base substrate of a microfluidic device as disclosed herein.

[0059] In various embodiments, silicon master molds can be created for a top substrate and for a base substrate of a microfluidic device as disclosed herein. In some embodiments, the master mold for a top substrate comprising a fluidic channel with a height of 80 jum can be made using a negative photoresist (SU-8 2050, Microchem, MA). In some embodiments, the master mold for a base substrate having one or more cavities can be fabricated using a two-step photolithography method. An exemplary procedure of a two-step photolithography method for fabrication of a base substrate having cavities with an upper and a lower surface is described as herein. In some embodiments, the first surface of the cavity (surface depths Hl= 20, 50, and 80 μπι) can be fabricated using negative photoresists (SU-8 2015, SU-8 2050). For example, for hl= about 20μπι, SU-8 2015 can be spin-coated at about 2,000 rpm for about 30 s, baked for about 3 min at about 95°C, and exposed to UV (140 mJ cm^" ) for about 1 min. As for a cavity with hi = about 50 or about 80μπι, they can fabricated by spin-coating SU-8 2050 at about 2,600 rpm and about 1,700 rpm for about 1 min, respectively. The second surface of the cavity at a height of h2 can be fabricated by spin coating SU-8 2050 photoresist on a silicon wafer patterned with the first surface patterns. Double-layer spin-coating processes enable deeper cavities with higher aspect ratios to be fabricated than single-step photolithography.

[0060] Following photolithography, negative replicas of the top substrate and bottom substrate can be molded in an elastomer by curing a mixture of an elastomer precursor and a curing agent. A variety of elastomer precursors and curing agents are commercially available, and one skilled in the art will be able to identify those suitable for use with embodiments of the inventions. Exemplary elastomers include, but are not limited to, silicone elastomers such as PDMS, acrylic elastomers such as VHB 4910, acrylic elastomer as produced by 3M Corporation of St. Paul, Minn., polyurethanes, thermoplastic elastomers, copolymers comprising PVDF, fluoroelastomers, polymers comprising silicone and acrylic moieties, and the like. In various embodiments, an elastomer such as poly(dimethyl siloxane) PDMS can be used to fabricate a base substrate and/or a top substrate. Other examples of substrate materials that can be used for the purpose of this invention include, but not limited to, glass, co-polymer or polymer.

Exemplary polymers include, but not limited to, urethanes, rubber, molded plastic,

polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, and the like. Further suitable materials include, but are not limited to, poly(ester amide), polystyrene- polyisobutylene-polystyrene block copolymer (SIS), polystyrene, polyisobutylene,

polycaprolactone (PCL), poly(L-lactide), poly(D,L-lactide), poly(lactides), polylactic acid (PLA), poly(lactide-co-glycolide), poly(glycolide), polyalkylene, polyfluoroalkylene, polyhydroxyalkanoate, poly(3-hydroxybutyrate), poly(4hydroxybutyrate), poly(3- hydroxy valerate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyhexanoate), poly(4-hyroxyhexanoate), mid-chain polyhydroxyalkanoate, poly (trimethylene carbonate), poly (ortho ester), polyphosphazenes, poly (phosphoester), poly( tyrosine derived arylates), poly( tyrosine derived carbonates), polyvinylidene fluoride (PVDF), polyhexafluoropropylene (HFP), polydimethylsiloxane, poly (vinylidene fluoride-cohexafluoropropylene) (PVDF-HFP), poly (vinylidene fluoride-co-chlorotrifluoroethylene) (PVDF-CTFE), poly(butyl methacrylate), poly(methyl methacrylate), poly(methacrylates), poly(vinyl acetate), poly(ethylene-co-vinyl acetate), poly(ethylene-co-vinyl alcohol), poly(ester urethanes), polyethers, polyethyleneglycols (PEGs), poly(ether-urethanes), poly(carbonate-urethanes), poly(silicone-urethanes), poly(urea- urethanes) and any combinations thereof.

[0061] As used herein, the term "curing agent" includes curing agents, crosslinking agents, gelling agents, etc. One exemplary curing agent includes, but not limited to, Sylgard 184 (Dow Corning Corporation). Generally, the elastomer base solution and the curing agent can be mixed in any ratio. In some embodiments, the elastomer precursor solution and the curing agent are mixed in a ratio of about 5:1 to about 50:1, from about 5:1 to about 25:1, from about 5:1 to about 20:1, or from about 7:1 to about 15:1.

[0062] The curing reaction can be carried out for any length of time sufficient for curing a particular elastomer chosen. Accordingly, in some embodiments, curing can last for at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 10 hours, or at least overnight.

[0063] Similarly, the curing reaction can be carried out at a temperature that is optimal for a particular elastomer chosen. Accordingly, in some embodiments, the curing temperature can be at least about 50°C, at least about 60°C, at least about 70°C, or at least about 75°C.

[0064] A skilled artisan will be readily able to identity a suitable elastomer material for use with embodiments of the inventions, and determine the time and temperature required for curing reaction of a selected ratio of an elastomer precursor solution to a curing agent. In one embodiment, a top substrate and a base substrate of the invention disclosed herein can be molded in poly(dimethylsiloxane) (PDMS) (Sylgard 184 Silicon elastomer, Dow Corning, MI) by curing a aboutl0:l mixture of silicon elastomer and a curing agent on the patterned silicon master at about 70°C for about 2 hours.

[0065] In some embodiments, a microfluidic device produced by the inventive methods described herein can be formed from any material comprising a solid material that comprises a solidified form of a hardenable liquid, and, in some embodiments, the structures can be injection molded or cast molded.

[0066] After curing, the molds, e.g., PDMS molds, are subsequently peeled off the silicon masters. The resulting top and base substrates can be used to produce a microfluidic device of the invention or they can be used as a master for the fabrication of additional top and base substrates using techniques known to a skilled artisan. In some embodiments, inlet and outlet ports for cell loading and cell culture medium perfusion can be created in the top substrate comprising a channel, e.g., using sharp punchers. Reservoirs, for example, of about 1mm to about 4mm in diameter or width, can be cut into inlets as well. For example, a reservoir is cut into a top substrate as demonstrated in Figure 1. In additional embodiments, a channel in a top substrate can be connected to a drain via tubing sealed in the outlet port. For example, metal tubing can be sealed into the outlet port with epoxy.

[0067] In some embodiments, the top and base substrates can then be aligned and irreversibly bonded using oxygen plasma (5 min at 30W, Harrick Scientific, NY). Although irreversible sealing can be desired for devices comprised of static microchannels, reversibly sealed microchannels have been shown to be useful applications such as surface patterning, in which a channel array is used to pattern a material on a substrate and then removed.

Accordingly, in some embodiments, the resulting top and base substrates can be reversibly bonded using the method disclosed in U.S. Patent App. No.: US 2007/0266801, which is incorporated herein in their entirety by reference. For example, a top substrate comprising one or an array of channels can form reversible sealing by simply pressurizing the top substrate against the base substrate. The pressure can either be positive or negative. For example, clamps or other like devices can be used to impart positive pressure, while reservoirs can be placed between the top substrate and the base substrate to contain a vacuum.

Cell capture, co-cultures and uses thereof

[0068] In further embodiments, the devices and methods of the present invention can be used for capturing cells on one or more surfaces within a cavity as disclosed herein and thus immobilizing cells from a flowing cell-containing fluid on the surfaces of the cavity. The method comprises (1) providing a microfluidic device as disclosed herein, and (2) flowing at least a first cell-containing fluid on an opening surface of at least one cavity of the microfluidic device, thereby at least one cell in the first cell-containing fluid is captured on one or more surfaces within the cavity of the microfluidic device.

[0069] In some embodiments, the first cell-containing fluid flowing on top of a surface having at least one cavity can be introduced with a flow rate Ql through a first inlet of one or more channels in the top substrate of the microfluidic device, wherein the one or more channels communicate with the cavity on the base substrate. In some embodiments, the first cell- containing fluid can comprise at least a first cell population.

[0070] In alternative embodiments, the first cell-containing fluid can comprise 2 or more different cell populations. The term "cell population" can comprise one or more cell types. The term "cell type" as used herein refers to a group of cells having a distinct set of morphological, biochemical and/ or functional characteristics (e.g., the ability to internalize a specific compound). The term "cell type" can refer, e.g., to a broad class of cells (e.g., cancer cells, non- cancer cells and nerve cells), a sub-generic class of cells (e.g., prostate cancer cells, HIV- infected cells and breast cancer cells), or a cell line.

[0071] In some embodiments, the cell can be selected from the group consisting of a mammalian cell, a reptilian cell, an avian cell, a fish cell, an insect cell, a fungal cell, a plant cell, a yeast cell, and a bacterial cell. Examples of mammalian cells include, but not limited to, HeLa cells (human); NIH3T3 cells (murine); embryonic stem cells; a cell type such as hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, and epithelial cells; HL-1 cardiomyocytes; Chinese hamster ovary (CHO) cells, Chinese hamster lung (CHL) cells, baby hamster kidney (BHK) cells, COS cells, THP cell lines, Jurkat cells, hybridoma cells, carcinoma cell lines, and the like. In one embodiment, the cell is HL-1 cardiomyocytes. Suitable cell lines can be comprised within e.g. the American Type Culture Collection and the German Collection of Microorganisms and Cell Cultures. In addition, cells which have been transfected with recombinant genes can also be used in the present invention. In some embodiments, mammalian cells comprise anchorage dependent cells, which can attach and spread onto surfaces.

[0072] Exemplary insect cell lines include, but are not limited to, Lepidoptera cell lines such as Spodoptera frugiperda cells (e.g. Sf9, Sf21) and Trichoplusia ni cells (e.g. High

FiveTM, BTI-Tn-5Bl-4).

[0073] Exemplary fungal cell include, but are not limited to, the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi. Representative groups of Ascomycota include, e.g., Neurospora, Eupenicillium (or Penicillium), Emericella (ox Aspergillus), Eurotium (ox Aspergillus), and the true yeasts listed above. Examples of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, e.g., saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicillium, Candida, and Altemaria. Representative groups of Zygomycota include, e.g., Rhizopus and Mucor. Fungal cells can also be yeast cells. Exemplary yeast cells include, but are not limited to,

ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti or Deuteromycota (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four sub-families, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces including S. pombe), Nadsonioideae, Lipomyooideae, and Saccharomycoideae (e.g., genera Pichia including P.

pastoris, P. guillermondii and P. methanollo ), Kluyveromyces including K. lactis, K. fragilis and Saccharomyces including S. carlsbergensis, S. cerevisiae, S. diastaticus, S. douglasii, S.

kluyveri, S. norbensis ox S. oviformis). The basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Other useful yeast host cells are Hansehula polymorpha, Yarrowia lipolytica, Ustilgo maylis.

[0074] Useful microorganism cells can be unicellular, e.g. a prokaryotes, or non- unicellular, e.g. eukaryotes. Useful unicellular cells can be Archeabacteria. Further useful unicellular cells can be aerobic bacterial cells such as gram positive bacteria including, but not limited to, the genera Bacillus, Sporolactobacillus, Sporocarcina, Filibacter, Caryophanum, Arthrobacter, Staphylococcus, Planococcus, Micrococcus, Mycobacterium, Nocardia,

Rhodococcus; ox gram negative bacteria including, but not limited to, the genera Acetobacter, Gluconobacter, Frateuria, Alcaligenes, Achromobacter, Deleya, Amoebobacter, Chromatium, Lamprobacter, Lamprocystis, Thiocapsa, Thiocystis, Thiodictyon, Thiopedia, Thiospirillum, Escherichia, Salmonella, Shigella, Erwinia, Enterobacter, Serratia, Legionella, Neisseria, Kingella, Eikenella, Simonsiella, Alysiella, Nitrobacter, Nitrospina, Nitrococcus, Nitrospira, Pseudomonas, Xanthomonas, Zoogloea, Fraturia, Rhizobium, Bradyrhizobium, Azorhizobium, Sinorhizobium, Rickettsia, Rochalimaea, Ehrlichia, Cowdria, Neorickettsia, Treponema, Borrelia, Vibrio, Aeromonas, Plesiomonas, Photobacterium, Brucella, Bordetella,

Flavobacterium, Francisella, Chromobacterium, Janthinobacterium, and lodobacter.

[0075] Suitable plant cells for use in the present invention can include dicotyledonous plant cells, examples of which are Arabidopsis Thaliana, tobacco, potato, tomato, and leguminous (e.g. bean, pea, soy, alfalfa) cells. Monocotyledonous plant cells, e.g.

monocotyledonous cereal plant cells such as for example rice, rye, barley and wheat, can be equally suitable.

[0076] A cell-containing fluid or a fluid, e.g., cell culture medium, can be flowed through at least one channel in a top substrate in communication with cavities on a base substrate by charging the inlet reservoirs with appropriate fluids and then drawing them through the channel using a syringe pump connected to the outlet by flexible, e.g., polyethylene, tubing. Alternatively, a cell-containing fluid or a fluid, e.g., cell culture medium can be flowed through a channel disposed within a top substrate by introducing it through an inlet reservoir with a positive pressure. Flow rates of a cell-containing fluid or a fluid can be achieved using various pumps, such as micromachined pumps, reciprocating pumps, peristaltic pumps, diaphragm pumps, syringe pumps, volume occlusion pumps as well as endoosmotic induced flow, and other pumping means known to those skilled in the art. In some embodiments, a syringe pump can be selected for the purpose of the invention. Alternative pumps can be microfabricated pumps, such as the HSG-IMT VAMP (Villengen, Germany) or other commercially available micropumps. In some embodiments, the pump can be incorporated into the device according to known microfabrication techniques.

[0077] Fluid flow through microfluidic devices can deliver nutrients, growth factors, and reagents to the cells captured inside. However, exposing the cells to flow can also lead to cell removal from the device. Therefore, an optimal inlet flow velocity U is desirable. In accordance with the invention, cell retention decreases approximately linearly with increasing inlet flow velocity U, which depends on flow rate Q. Accordingly, U can be adjusted by changing the flow rate Q. In some embodiments, Q can have a range from about 0.1 to about 1000 uL/min, about 0.5 to about 500 uL/min, or about 1 to about 100 uL/min. In one embodiment, Q can range from about 5 to about 40 uL/min. In further embodiment, Q can be further optimized for each cell population that exhibit different binding affinity to a substrate. In one embodiment, for cells that are less adherent to a cavity surface, a smaller Q can be applied.

[0078] The flow can be stopped for a few minutes, for example, between about 5 and about 20 minutes, to allow cells forming adhesions to one or more surfaces within at least one cavity of a microfluidic device as disclosed herein. One skilled in the art will recognize that the flow rate, cell concentration, and flow stop time can all be optimized to capture a desired number of cells into each cavity or various surfaces at different depths within each cavity, and allow the cells to become immobilized by adhesion to the base substrate. Excess cells can be removed by applying a suction force to the inlet reservoir, e.g., aspirating the cells from the inlet reservoir with a pipette, or flowing a cell-free fluid, e.g., cell culture medium or phosphate buffered saline at a higher flow rate Q through the channel to remove less adhesive cells.

[0079] In various embodiments, a cell adhesive material can be deposited in the cavities, for example, using robotic techniques, microfluidic techniques, photolithographic techniques, or microstamping techniques. For example, a stamp can be used to deposit a cell and protein resistant coating on the surface of the substrate, following which the cavities can be coated with a cell adhesive proteins. Such proteins include, for example, fibrinogen, collagen, laminin, integrins, antibodies, antigens, cell receptor proteins, cell receptor antagonists, and mixtures of the above.

[0080] In another embodiment of the invention wherein the cell-containing fluid is blood, plasma, or a solution containing blood products likely to cause coagulation such as platelets, the cavities in the base substrate and channels in top substrate can be treated to inhibit coagulation and clot formation. Examples of such anti-coagulant surface treatments which are known in the art to prevent coagulation and clot formation include heparin, heparin fragments, tissue-type plasminogen activator (tPA), urokinase (uPA), anti-thrombosis agents (such as Hirudan) and albumin. Also suitable are anti-coagulant agents which can be antibodies, for example antibodies directed against platelet receptor GPIB and/or GPIB, against platelet receptor GPIIb/IIIa, and/or against von Willebrand Factor (vWF).

[0081] The position of cells on one or more surfaces within at least one cavity of a microfluidic device as disclosed herein can be moved from downstream surfaces to upstream surfaces by adjusting the ratio of h2 to hi. In accordance with the invention, the geometry of the cavity can create different flow regimes, such as no eddy, single eddy, or double eddy, which direct the cells to a specific position. When the ratio of h2 to hi is about 1 or any value greater than 1, a counter-rotating eddy can be generated on the lower surface and the cells can be captured upstream. In one embodiment, the value can be an integer, e.g., about 1, about 2, about 3, about 4, about 5, or about 6. In another embodiment, the value can include an integer plus a fraction, e.g., any continuum values from about 1 and beyond, e.g., about 4.1, about 4.2 or about 4.3. Further, in accordance with the invention, when the ratio of h2 to hi is any value less than 1, the cells can be captured downstream. In one embodiment, the ratio of h2 to hi can be any fraction, e.g., any continuum values less than 1, e.g. about 0.1, about 0.2, about 0.25, about 0.3, about 0.4 or about 0.5. Accordingly, the capture position of the cells within the cavity can be moved horizontally upstream, when the ratio of h2 to hi is adjusted from a value smaller than 1 to a value of about 1 to a value greater than 1. Similarly, the capture position of the cells within the cavity can move horizontally downstream, when the ratio of h2 to hi is adjusted from a value greater than 1 to a value of about 1 to a value smaller than 1. Further, the positions of cells on surfaces within the cavities can be controlled by adjusting the ratio of hO/il or (hl+h2)/[l. In some embodiments, the positions of cells on surfaces within the cavities can be controlled by adjusting one or more ratios selected from the group consisting of: hO/il, (hl+h2)/[l or h2/hl, as

demonstrated by the method disclosed in Example 4.

[0082] In various embodiments, the cell position on one or more surfaces of a cavity depends on the ratio of h2 to hi and other aspect ratios (e.g., hO/il or (hl+h2)/[l). For example, as shown in the phase diagrams of Figures 6 A to 6E or discussion in Example 4, depending on the flow condition (e.g. Re) and other aspect ratios (hO/il or (hl+h2)/[i)), the ratio of h2 to hi can be smaller than 1 for upstream capture of cells. Similarly, the ratio of h2 to hi can be greater than 1 for downstream capture of cells.

[0083] In accordance with the invention, cell position on the surfaces of the cavity can be influenced by the flow pattern, which in turn is determined by the flow conditions (e.g., Reynolds' number) and the overall predefined geometry of the cavity, such as determined by other aspect ratios, e.g., hO/il, (hl+h2)/[l. Accordingly, the phase diagrams as disclosed in Figures 6A to 6E can be used to map out different system flow behaviors for a cavity having an upper and a lower surface (e.g., to determine critical values of h2/hl that separate downstream cell-capture from upstream cell-capture) and thus determine the optimal cavity geometry. One of skill in the art will be able to generate such phase diagrams for a cavity having more than 2 surfaces, using the methods disclosed in Examples 2 and 4, as well as to determine the optimal geometry for the desired cell position and capture. [0084] In accordance with the invention, the cells accumulate on the low- shear surfaces and/or corners of a cavity disposed with a microfluidic device. The position of the surfaces and/or corners of the cavity in that they accumulate depends on the flow direction and flow regime (e.g., no eddy, single eddy, or double eddy), which in turn depends on the geometry and/or aspect ratios of the cavity. Once the flow is stopped, the cells can settle and can attach to the surface and be immobilized (e.g. if the capture surface is coated with cell adhesion molecules or agents). Accordingly, in further embodiments, by adding a flow control system (e.g., with a microscope and/or camera), a single cell can be positioned anywhere on the surfaces of the cavity by monitoring the cell's trajectory, and then stopping the flow at just the right time so it would fall at the desired location. For example, in general, cells unlikely accumulate at the convex corners of a protruding object (e.g., a step) within a cavity without precise flow control, because the flow speed and shear stress there are both high. However, with a flow control system (e.g., with a microscope), when a cell flows to a point above the convex corner (e.g., a ledge), and the flow is stopped, the cell can be positioned at the ledge. Accordingly, with advancement of technology, e.g, precise flow control system or a system for monitoring a cell's trajectory (e.g, with a camera and/or microscope), the position of at least one cell on one or more surfaces of the cavity can be controlled.

[0085] In accordance with the invention, as h2 increases, there is less positioning control on the lower surface. Additionally, when h2 is smaller than a cell diameter, or close to about a cell diameter, e.g., about 5um, about lOum, or about 20um, or about 30um, it can be more difficult to positions cells on the lower surface. Accordingly, in some embodiments, there can be a desirable range of values for h2. One of skill in the art can determine the optimal value of h2 for a specific purpose by computational analysis, e.g., using the method disclosed in Example 2.

[0086] In additional embodiments, the method of the invention further comprises flowing at least a second fluid at a flow rate Q2 through a second inlet of a second channel in the top substrate of the microfluidic device. In some embodiments, the second inlet of the second channel can be same as the first inlet of the first channel disposed within the top substrate of the invention. In alternative embodiments, the second inlet of the second channel can be the opposite end of the first channel. In further embodiments, the second inlet of the second channel can be different from the first inlet of the first channel.

[0087] Similarly, the second fluid can be the first cell-containing fluid. In circumstances where higher number of cells to be captured is desirable, the flow can be repeated at a flow rate Q2, which is smaller than Ql, through the same previous inlet. Alternatively, the first cell- containing fluid can be introduced from the other end of the first channel, or through a second channel that communicates with the cavity.

[0088] In other embodiments, the second fluid can be cell-free. For instance, after cell capture, the cell-containing fluid can be switched to cell culture medium for removing any nonadherent cells and/or maintaining the cells in culture. The cell culture medium can be flowed (e.g., Q2 equal to Ql) through the same previous channel or a new channel. In other

embodiments, the flow can be stopped once the cavities are filled with cell culture medium. The volume of cell culture media in the cavities can range from about lOnL to about 500nL. In some embodiments, a cavity can have from about lOnL to about lOOnL of cell culture media. The volume of cell media required in each cavity depends on the cavity capacity. A skilled artisan will be able to adjust the volume of cell culture media accordingly.

[0089] In yet another embodiment, test compounds can be optionally added to cell culture medium for cell-based biosensing or drug screening, which will be discussed further later. In some embodiments, cell culture medium can be flowed at Q2 greater than Ql to remove cells that have already attached to the base substrate. Accordingly, based on the cell condition and experimental outcome, the flow rate of the second fluid (Q2) can be adjusted to achieve desirable cell capture. In some embodiments, Q2 can be greater than Ql. In some embodiments, Q2 can be smaller than Ql. In other embodiments, Q2 can be equal to Ql.

[0090] In further embodiments, the second fluid can contain one or more distinct cell types from the first cell-containing fluid. In accordance with the invention, aspect ratios of a cavity geometry determines whether cells immobilize upstream or downstream (Figure 4).

Without wishing to be bound by theory, by reversing the flow direction of a cell-containing fluid, the location of cell capture becomes the reverse.

[0091] Accordingly, another aspect of the invention provides methods for co-culturing at least two cell populations in a microfluidic device. Different cell types can be separated and captured at distinct locations of a cavity by changing the direction of the flow. Without wishing to be bound by theory, for example, when the cavity is of a square or a rectangular shape, one distinct cell population can be positioned or captured on each side of the cavity. For example, in one embodiment, the first cell-containing fluid with a first cell population can be flowed through a first channel in one direction, and the cells can be captured on one side of the cavity surfaces (e.g. upstream or downstream surfaces of the cavity). When the second cell-containing fluid with a second cell population can be flowed through the first channel in the opposite direction, e.g., by introducing the second cell-containing fluid from the other end of the first channel, the second cell population can be captured on the opposite side of the cavity surfaces. Additionally, when the third cell-containing fluid with a third cell population can be flowed through a second channel that is orthogonal to the first channel, the third cell population can be captured on the surfaces of the cavity orthogonal to the first and/or second cell populations captured previously within the cavity. Similarly, the fourth cell-containing fluid with a fourth cell population can be flowed through the second channel in the opposite direction, e.g., by introducing the fourth cell- containing fluid from the other end of the second channel, allowing the fourth cell population to be captured on the surfaces opposite to the surfaces on which the third cell population are captured. Accordingly, the maximum number of distinct cell populations that can be captured or positioned on different portion of the surfaces within a cavity can be controlled by the shape of a cavity, e.g. a square, a rectangle, a polygon, the flow direction, and/or cavity dimensions. In such embodiments, orthogonal flows, the reversed flows or a combination thereof can be applied.

[0092] In other embodiments, two or more different cell types can be captured separately in a cavity, as described above, and can be allowed to grow upon the surface and spread such that cells of the two or more different cells types spread together and come into contact on the surface after a period of time has elapsed. Such a co-culture method can be useful as part of an in vitro assay, which is able to determine and/or study interactions between different cell types. For example, such method can form part of an in vitro assay able to determine an angiogenic potential of a particular type of tumor cell. In one particular application, two different cell types comprising capillary endothelial cells and tumor cells can be each captured on one side of a cavity and allowed to grow and spread upon the surface after capture, as described above, in order to simulate and study angiogenesis during tumor formation. In an in vivo model, tumor cells tend to attract and direct the growth of capillary endothelial cells to form new blood vessels to supply nutrients and oxygen for tumor growth. By forming a defined pattern of capillary endothelial cells and tumor cells utilizing the devices and methods provided according to the invention, it can be possible to enable assays able to study the differential and competitive attraction of capillary endothelial cells to different tumor cell lines. This technique, enabled by the present invention, can lead to the development of a simple, standardized, and quantitative in vitro assay for comparing the angiogenic potential of different tumor cells.

[0093] In alternative embodiments, a co-culture of two or more different cell types in contact with each other can be generated. In such embodiments, the first cell-containing fluid can comprise more than one cell types. In another embodiment, the first cell-containing fluid can comprise one cell type and the second cell-containing fluid can comprise other different cell types. In addition, both the first and second cell-containing fluids can have the same flow direction through a channel disposed within a top substrate of a microfluidic device. Such a co- culture method can be another useful in vitro assay, which is able to determine and/or study cell- cell interactions between two different cell types.

[0094] In some embodiments, at least one cavity on the microfluidic device of the invention comprises one or more cells. It is to be understood that number of cells captured on each surface the cavity will depend on a function of binding affinity of cells to substrates, cell retention time, flow rate of a fluid passing through the channel in the top substrate and the cavity dimensions. Typically, a cavity can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 or more cells. In some embodiments, a cavity can comprise at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, or at least 100 or more cells. In some embodiments, about 5 to about 10 cells can be captured in a cavity. In some embodiments, about 10 to about 500 cells can be captured in a cavity. In one embodiment, about 10 to about 100 cells can be captured in a cavity.

[0095] A skilled artisan can adjust the cell seeding density of the cell-containing fluid to adjust the number of cells captured in a cavity. Accordingly, in some embodiments, the cell seeding density can be about 10 3 to about 109 cells, about 104 to about 108 cells, or about 105 to about 10⁷ cells. In one embodiment, the cell seeding density can be about 10⁶ cells.

[0096] The cells captured in a cavity can be present as a monolayer, or an aggregate. As used herein, the terms "monolayer" refer to cells that have adhered to a substrate and grow in as a layer that is one cell in thickness. The term "aggregate" as used herein refers to a group of cells growing on a layer that is one cell in thickness, or a group of cells growing in layers.

[0097] In accordance with the invention, cells suspended in a flowing fluid can be immobilized upstream in a deep and narrow cavity, and can be immobilized downstream in a shallow and wide cavity (Figure 4). Accordingly, in some embodiments of the invention, at least one cell can be captured on at least a first surface and a second surface of a cavity of a microfluidic device as disclosed herein. In other embodiments, a cell can be captured on upstream portion of a first and/or second surface of the microfluidic device. In alternative embodiments, a cell can be captured on downstream portion of a first and/or a second surface of the microfluidic device.

[0098] Channels disposed within a top substrate of a microfluidic device can be used to flow different combinations of nutrients to cells in various rows or columns of cavities. For example, different groups of cells can be provided with cell culture media that can be deficient in a particular nutrient or to which a particular nutrient has been added.

[0099] The invention can be used at any biologically viable temperature. Lowering the incubation temperature of the cells (e.g., from 37° C. to 18° C.) can slow the metabolic processes of the cells and the cell doubling time, thus extending the time for individual cell experimentation and manipulation.

Uses of a microfluidic device of the invention

[00100] Another aspect of the invention provides microfluidic devices and methods for cell separation. In accordance with the invention, the shear stress on the upper surface of the cavity as disclosed herein is generally higher than the shear stress on the lower surface of the cavity. In one embodiment, cells with a higher cell adhesion affinity to the surface can be captured on the upper surface, but not cells with weak cell adhesion affinity. In such

embodiments, the cells with a higher cell adhesion affinity can be isolated from a cell-containing fluid comprising more than one cell types.

[00101] Still another aspect of the invention provides microfluidic devices and methods for cell-based biosensing and drug screening. Once cells are captured, a variety of chemicals can be flowed through the channels to modify the metabolism, membrane characteristics, contrast properties, or other properties of the cells. For example, solutions containing a targeting agent can be flowed through the channels. Targeting agents can include any growth factor, antibody, cytokine, ligand, small molecule, bioactive agent, or biomolecule, natural or synthetic, that binds specifically to a cell surface receptor, protein or glycoprotein found at the surface of cells.

Targeting agents can include but are not limited to antibodies and antibody fragments, nucleic acid ligands (e.g., aptamers), oligonucleotides, oligopeptides, polysaccharides, low-density lipoproteins (LDLs), folate, transferrin, asialycoproteins, gpl20 envelope protein of the human immunodeficiency virus (HIV), carbohydrates, polysaccharides, enzymatic receptor ligands, sialic acid, glycoprotein, lipid, small molecule, bioactive agent, biomolecule, immunoreactive fragments such as the Fab, Fab', or F(ab¾ fragments, etc. A variety of targeting agents that direct pharmaceutical compositions to particular cells are known in the art (see, for example, Cotton, et al., Methods Enzym. 217:618; 1993; incorporated herein by reference).

[00102] Exemplary growth factors include, but are not limited to, activin A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, platelet derived growth factor, hepatocyte growth factor, insulin-like growth factors (IGF) I and II, hematopoietic growth factors, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony stimulating factors, heparin binding growth factor (HBGF), alpha or beta transforming growth factor (a- or β-TGF), fibroblastic growth factors, epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), nerve growth factor (NGF) and muscle morphogenic factor (MMP).

[00103] Solutions containing one or more contrast agents can be flowed through the channels. These contrast agents can be used to stain cells that exhibit certain surface proteins or that are producing particular materials. On a more basic level, they can be used to identify viable cells. Suitable contrast agents are well known to those of skill in the art and include, but are not limited to, fluorescent markers, radionuclides, and cellular dyes. Additional contrast reagents include visible dyes, fluorescent dyes and radioactive dyes. Such dyes can react directly with components produced by the cells in reaction to the candidate compound. Alternatively, such dye can be bound either covalently or non-covalently to a ligand or antibody which binds to components produced by the cells in reaction to the candidate compound. Radioactive dyes include ³²P, ¹²⁵I and ³H. Fluorescent dyes include fluorescein CALCEIN-AM, FLUO-3, FURA- 2, INDO-1 QUIN-2 and related compounds available from Molecular Probes. Fluorescent pH indicators include compounds such as SNAFL, SNARF and related pH indicators. Cell viability can be measured using the compound, CALCEIN-AM. DNA can be detected in dead cells with ethidium homodimer.

[00104] A microfluidic device of the invention can be used to screen potential pharmaceutical agents. In some embodiments, co-cultures generated by the methods of the invention can provide an in vitro model for cell-based biosensing and drug screening, as many diseases such as cancer involves more than one cell type. Examples of pharmaceutical agents include, but not limited to, anti-AIDS substances, anti-cancer substances, antibiotics, immunosuppressants, anti-viral substances, enzyme inhibitors, including but not limited to protease and reverse transcriptase inhibitors, fusion inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, ranquilizers, anti-convulsants, muscle relaxants and anti Parkinson substances, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti- adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, antihypertensives, analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-secretory factors, anticoagulants and/or antithrombotic agents, local anesthetics, ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti- emetics, and imaging agents. In various embodiments, the pharmaceutical agent can be a drug. A more complete listing of specific drugs suitable for use in the present invention can be found in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", Edited by Susan Budavari et al., CRC Press, 1996, and the United States Pharmacopeia-25/National Formulary-20, published by the United States Pharmcopeial Convention, Inc., Rockville Md., 2001, all of which are incorporated herein by reference.

[00105] The microfluidic device can also be utilized in combination with an appliance for viewing the effect of the candidate compound on the cells. The appliance in one embodiment can comprise a microscope for viewing the effect. The microscope can be an inverted microscope or a confocal microscope. A UV light source or laser beam used to activate cell fluorescence can also be used to observe the effects of candidate compounds on a cell.

[00106] The present invention may be defined in any of the following numbered paragraphs:

1 . A microfluidic device comprising:

a base substrate having a top surface and at least one cavity in the top surface, the cavity comprising at least a first surface and a second surface, wherein:

the first surface is located at a predefined distance Hi below the top surface; and the second surface is located at a predefined distance H2 below the first surface.

2. The microfluidic device of paragraph 1, further comprising a top substrate mounted on the top surface of the base substrate, the top substrate defining at least one channel extending over the base substrate, wherein the at least one channel is in communication with the cavity on the base substrate and the at least one channel has a predefined height HO.

3. The microfluidic device of paragraph 1, wherein the first surface and the second surface are concentric.

4. The microfluidic device of paragraph 1, wherein the first surface has a center and the second surface has a center and the center of the first surface is not aligned with the center of the second surface.

5. The microfluidic device of paragraph 1, wherein the first surface or the second surface is asymmetric with respect to an axis transverse to the at least one channel.

6. The microfluidic device of paragraph 1, wherein at least one of the first surface and the second surface have an irregular shape. The microfluidic device of paragraph 1, wherein at least one of the first surface and the second surface have a regular shape.

The microfluidic device of paragraph 7, wherein the regular shape is selected from a group comprising a circle, a square, a rectangle, a triangle, a trapezoid, and a polygon. The microfluidic device of any of paragraphs 1 to 8, wherein the first surface has a predefined width il and the second surface has a predefined width [2, wherein il, [2 are determined as a function of hO, hi, h2, a desired number of cells on each of the first and the second surfaces, corner eddies, a shear stress profile in the cavity, and properties of fluid.

The microfluidic device of paragraph 9, wherein the desired number of cells on each of the first and the second surfaces is determined as a function of binding affinity of cells to the base substrate, cell retention time, flow rate of a fluid passing through the channel in the top substrate and area of the first and the second surfaces.

The microfluidic device of paragraphs 2 and 9, wherein hO is determined as a function of il and properties of fluid.

The microfluidic device of paragraphs 9 and 11, wherein the properties of fluid comprise a flow rate of a fluid passing through the channel in the top substrate, and a density and viscosity of the fluid.

The microfluidic device of any of paragraphs 1 to 12, wherein the ratio of hO to il is less than 1.

The microfluidic device of any of paragraphs 1 to 12, wherein the ratio of hO to il is in the range of about 0.2 to about 0.7.

The microfluidic device of any of paragraphs 1 to 12, wherein il is greater than [2.

The microfluidic device of any of paragraphs 1 to 12, wherein the ratio of il to [2 is about 2.

The microfluidic device of any of paragraphs 1 to 12, wherein hi and h2 are each independently greater than a cell diameter.

The microfluidic device of any of paragraphs 1 to 12, wherein hi is at least about ΙΟμπι. The microfluidic device of any of paragraphs 1 to 12, wherein hi is at least about 20μπι. The microfluidic device of any of paragraphs 1 to 12, wherein hi is at least about 30μπι. The microfluidic device of any of paragraphs 1 to 12, wherein h2 is at least about ΙΟμπι. The microfluidic device of any of paragraphs 1 to 12, wherein h2 is at least about 20μπι. The microfluidic device of any of paragraphs 1 to 12, wherein h2 is at least about 30μπι. The microfluidic device of any of paragraphs 1 to 12, wherein the ratio of h2 to hi is any value greater than 1.

The microfluidic device of any of paragraphs 1 to 12, wherein the ratio of h2 to hi is equal to about 1.

The microfluidic device of any of paragraphs 1 to 12, wherein the ratio of h2 to hi is any value less than 1.

A method of making a microfluidic device, the method comprising:

providing a base substrate having a top surface;

forming at least one cavity in the top surface, the cavity comprising at least a first surface and a second surface;

forming the first surface at a predefined distance hi below the top surface;

forming the second surface at a predefined distance h2 below the first surface; and mounting a top substrate on the top surface of the base substrate, the top substrate including at least one channel in communication with the cavity on the base substrate, the channel having a predefined height ho.

The method of paragraph 27, wherein the first surface and the second surface are concentric.

The method of paragraph 27, wherein the first surface and the second surface are asymmetric.

The method of paragraph 27, wherein the first surface or the second surface is asymmetric.

The method of paragraph 27, wherein at least one of the first surface and the second surface have an irregular shape.

The method of paragraph 27, wherein at least one of the first surface and the second surface have a regular shape.

The method of paragraph 32, wherein the regular shape is selected from a group comprising a circle, a square, a rectangle, a triangle, a trapezoid, a polygon and the like. The method of any of paragraphs 27 to 33, wherein the first surface has a predefined width il and the second surface has a predefined width [2, wherein [1 and [2 are determined as a function of ho, hi, h2, a desired number of cells on each of the first and the second surfaces, corner eddies, a shear stress profile in the cavity, and properties of fluid.

The method of paragraph 34, wherein the desired number of cells on each of the first and second surfaces is determined as a function of binding affinity of cells to the base substrate, cell retention time and flow rate of the fluid passing through the channel in the top substrate.

The method of paragraphs 27 and 34, wherein hO is determined as a function of il and properties of fluid.

The method of paragraphs 34 and 36, wherein the properties of the fluid comprise a flow rate of the fluid passing through the channel in the top substrate, and a density and viscosity of the fluid.

The method of any of paragraphs 27 to 37, wherein the ratio of hO to il is less than 1.

The method of any of paragraphs 27 to 37, wherein the ratio of hO to il is in the range of about 0.2 to about 0.7.

The method of any of paragraphs 27 to 37, wherein il is greater than [2.

The method of any of paragraphs 27 to 37, wherein the ratio of il to 12 is about 2.

The method of any of paragraphs 27 to 37, wherein hi and h2 are each independently greater than about a cell diameter

The method of any of paragraphs 27 to 37, wherein hi is at least about ΙΟμπι.

The method of any of paragraphs 27 to 37, wherein hi is at least about 20μπι.

The method of any of paragraphs 27 to 37, wherein hi is at least about 30μπι.

The method of any of paragraphs 27 to 37, wherein h2 is at least about ΙΟμπι.

The method of any of paragraphs 27 to 37, wherein h2 is at least about 20μπι.

The method of any of paragraphs 27 to 37, wherein h2 is at least about 30μπι.

The method of any of paragraphs 27 to 37, wherein the ratio of h2 to hi is greater than 1.

The method of any of paragraphs 27 to 37, wherein the ratio of h2 to hi is equal to about 1.

The method of any of paragraphs 27 to 37, wherein the ratio of h2 to hi is less than 1. A method for capturing at least one cell in at least one cavity of a microfluidic device, the method comprising:

providing a microfluidic device of paragraph 2; and

flowing at least a first cell-containing fluid into a first inlet of the microfluidic device, whereby at least one cell from the first cell-containing fluid is captured in the cavity of the microfluidic device.

The method of paragraph 52, wherein the first cell-containing fluid flows at a flow rate Ql through the at least one channel.

The method of paragraphs 52 and 53, wherein the first cell-containing fluid comprises at least a first cell population. 55. The method of any of paragraphs 52 to 54, wherein the first cell-containing fluid further comprises at least a second cell population.

56. The method of paragraph 52, wherein at least one cell is captured on the first surface of the cavity.

57. The method of paragraph 52, wherein at least one cell is captured on the second surface of the cavity.

58. The method of paragraph 52, wherein at least one cell is captured on an upstream portion of the first surface of the cavity.

59. The method of paragraph 52, wherein at least one cell is captured on an upstream portion of the second surface of the cavity.

60. The method of paragraph 52, wherein at least one cell is captured on a downstream

portion of the first surface of the cavity.

61. The method of paragraph 52, wherein at least one cell is captured on a downstream

portion of the second surface of the cavity.

62. The method of any of paragraphs 56 to 61, wherein a position of the cells captured on the first surface or the second surface can be controlled as a function of a ratio of h2 to hi.

63. The method of paragraph 62, wherein the ratio of h2 to hi is greater than 1.

64. The method of paragraph 62, wherein the ratio of h2 to hi is equal to about 1.

65. The method of paragraph 62, wherein the ratio of h2 to hi is smaller than 1.

66. The method of paragraph 52, further comprising flowing at least a second fluid at a flow rate Q2 through a second inlet of at least a second channel in the top substrate of the microfluidic device.

67. The method of paragraph 66, wherein the second fluid is same as the first cell-containing fluid.

68. The method of paragraph 66, wherein the second fluid is cell-free.

69. The method of paragraph 66, wherein the second fluid contains at least a third cell

population.

70. The method of paragraph 55 wherein the first and second cell populations are

prokaryotic cells.

71. The method of paragraph 69, wherein the third cell population is prokaryotic cells.

72. The method of paragraph 55 wherein the first and second cell populations are eukaryotic cells.

73. The method of paragraph 69, wherein the third cell population is eukaryotic cells.

74. The method of any of paragraphs 72 to 73, wherein the eukaryotic cells are mammalian cells. 75. The method of any of paragraphs 52 to 74, wherein Ql is equal to Q2.

76. The method of any of paragraphs 52 to 74, wherein Ql is greater than Q2.

77. The method of any of paragraphs 52 to 74, wherein Ql is smaller than Q2.

78. The method of any of paragraphs 52 to 77, further comprising

providing a flow control system and using the flow control system to monitor a cells trajectory to control a capture position of the cell on the first surface or the second surface.

79. The method of any of paragraphs 52 to 78, wherein the flow is laminar flow.

80. A method of co-culturing at least two cell types in a microfluidic device, the method comprising:

providing a microfluidic device of paragraph 2; and

flowing at least a first fluid containing at least two cell types into a first inlet of the microfluidic device, whereby at least two cell types in the first fluid are captured in the cavity of the microfluidic device.

81. The method of paragraph 80, wherein the first fluid flows at a flow rate Ql through the at least a one channel.

82. The method of paragraph 80, further comprising flowing at least a second fluid at a flow rate Q2 through a second inlet of at least a second channel in the top substrate of the microfluidic device.

83. The method of paragraph 80, wherein the first fluid comprises at least a first cell

population.

84. The method of paragraph 80, wherein the first fluid further comprises two or more cell populations.

85. The method of paragraph 82, wherein the second fluid is same as the first fluid.

86. The method of paragraph 82, wherein the second fluid is cell-free.

87. The method of paragraph 82, wherein the second fluid comprises at least a third cell type.

88. The method of paragraphs 80 and 87, wherein the first, second and third cell types are prokaryotic cells.

89. The method of paragraphs 80 and 87, wherein the first, second and third cell types are eukaryotic cells.

90. The method of paragraph 89, wherein the eukaryotic cells are mammalian cells.

91. The method of any of paragraphs 80 to 90, wherein at least one cell is captured on a first surface of the cavity. 92. The method of any of paragraphs 80 to 90, wherein at least one cell is captured on a second surface of the cavity.

93. The method of any of paragraphs 80 to 90, wherein at least one cell is captured on an upstream portion of the first surface.

94. The method of any of paragraphs 80 to 90, wherein at least one cell is captured on an upstream portion of the second surface.

95. The method of any of paragraphs 80 to 90, wherein at least one cell is captured on a downstream portion of the first surface.

96. The method of any of paragraphs 80 to 90, wherein at least one cell is captured on a downstream portion of the second surface.

97. The method of any of paragraphs 80 to 96, wherein a position of the cells captured on the first surface or the second surface can be controlled as a function of a ratio of h2 to hi.

98. The method of paragraph 97, wherein the ratio of h2 to hi is greater than 1.

99. The method of paragraph 97, wherein the ratio of h2 to hi is equal to about 1.

100. The method of paragraph 97, wherein the ratio of h2 to hi is smaller than 1.

101. The method of paragraph 82, wherein the second channel is orthogonal to the at least one channel.

102. The method of any of paragraphs 80 to 101, wherein Ql is equal to Q2.

103. The method of any of paragraphs 80 to 101, wherein Ql is greater than Q2.

104. The method of any of paragraphs 80 to 101, wherein Ql is smaller than Q2.

105. The method of any of paragraphs 80 to 104, further comprising

106. The method of any of paragraphs 80 to 105, wherein the flow is laminar flow.

EXAMPLES

[00107] The following Examples are intended to further illustrate certain embodiments of the invention and are not to be construed as limiting the scope of the invention.

[00108] The examples presented herein relate to microfluidic devices, methods for design and fabrication of the microfludic devices, as well as methods for immobilizing cells suspended in a flowing fluid. Throughout this application, various publications are referenced. The disclosures of all of the publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods which occur to the skilled artisan are intended to fall within the scope of the present invention.

Materials and Methods

[00109] Cell capture and retention in double surfaces: A cardiac muscle cell line (HL- 1) derived from the AT-1 mouse atrial cardiomyocyte tumor was used to study cell capture in the double microsurfaces in our microfluidic device. HL-1 cells were cultured with medium in a humidified incubator (37 °C, 5% C02). The cell culture medium consisted of 87% Claycomb Medium, 10% Fetal Bovine Serum (FBS), 1% L-Glutamine, 1% Norepinephrine, and 1% Penicillin/Streptomycin. Cells were trypsinized and dissociated with the culture medium and then seeded at a concentration of 4 X 10⁶ cells/mL through the inlet port in the microfluidic device. After 10 minutes of cell seeding, culture medium was infused through the inlet port using a syringe pump at flow rates Q ranging from 5 to 40 μΐνηιίη. These flow rates generated average inlet velocities U = Q/(zi>0 ho) ranging from 0.052 to 0.417 cm/s (Table 3). For cell- capture experiments, a constant flow rate of Q= 5 μΐνηιίη was used. For the cell retention experiments, cells were captured on one or more surfaces within cavities of a microfluidic device as disclosed herein, and then exposed to the various inlet velocities listed above. Cell capture and retention for the six different substrate design configurations with various combinations of cavity aspect ratios listed in Table 1 in Example 1 were analyzed. For the cell- capture and retention experiments, the flow speed was held constant until steady state conditions were reached, and then images of cells in the cavities were obtained using an inverted microscope (Nikon TE 2000-U, USA). For cell-capture experiments, cells in the four surface corners were counted for each cavity geometry. The experiments were repeated three times for each flow condition. For cell-retention experiments, cells on each surface were counted; the experiments were repeated three times.

[00110] Dimensional analysis and assumptions: For the purpose of modeling, it was assumed that the flow in the microchannel and on surfaces is steady and 2D. The former assumption holds since the flow rates in the experimental setup are steady. The latter assumption is valid since their channel width is roughly ten times its height and the surface lengths are roughly ten times their surface widths and heights. 2D flow in cavities having an upper and a lower surface is characterized by 9 physical input parameters: 6 specify the geometry, i.e., inlet channel width τνθ and height ho, upper and lower surface widths [1, [2 and heights hi, h2 (Table 1 in Example 1 and Figure 2C), and 3 specify the fluid, i.e., the flow rate Q (cm /s) and the density p and kinematic viscosity v of the culture medium, taken to be that of water [24] at 20 °C, p = 1.0 g cm^"3 and v = 0.01cm² /s.

[00111] Dimensional analysis reduces the dependence of the 2D flow in such cavities of the invention from 9 physical input parameters to 5 dimensionless parameters: 4 aspect ratios ΙΪΟΙίΙ, ΙΪ2ΙΙΪ1, (ΙΪ1+ΙΪ2)Ι (1, and (2/(1 (Table 2 in Example 2) and the Reynolds number Re =

Q/(wo v) of the inlet channel. For comparison, the flow in a single-surfaced channel is completely described by 3 dimensionless parameters, hold, hllil and Re. In the experiments, the Reynolds number Re can range from 0.04 to 0.33 (Table 3 in Example 2). At these small Reynolds numbers, the flow is laminar and close to the Stokes flow limit, where the scaled velocities, shear stresses, and pressure gradients can be purely dependent on geometry and not on the flow rate [25]. Introducing and working in dimensionless co-ordinates not only reduces the apparent complexity of the problem by half but also elucidates the flow regime and simplifies the dependencies expected in the numerical simulations.

[00112] Governing equations: The Navier-Stokes equations, which consist of the incompressibility condition and the conservation of momentum, govern the fluid flow. All spatial variables were scaled by the width (1 of the upper surface and flow velocities were scaled by the inlet velocity U¼Q/(H;0 HO) and pressure by pvU/fl. In dimensionless variables, the steady Navier-Stokes equations become [26]

where v = (u, v) is the 2D velocity vector, u, υ are the horizontal (longitudinal, x-direction) and vertical (y-direction) velocity components, p is the modified pressure due to dynamic effects

[25], V the gradient operator and V the Laplacian operator. The no-slip boundary condition is applied at the top and bottom walls. The velocity profile in the inlet channel is assumed to be fully developed Poiseuille flow. For the low Reynolds numbers in the parameter regime of interest, the flow in the inlet channel becomes fully developed over a distance lio away from the ports and surfaces [27] . The Dirichlet boundary condition u=u0, υ =0 were imposed at the inlet, where the scaled inlet velocity uO has the Poiseuille profile

and y is the dimensionless vertical coordinate. The modified pressure is set to zero at the outflow boundary (Dirichlet boundary condition).

[00113] Numerical simulation and domain discretization: In the present work, the commercial finite element package Comsol 3.4 (Comsol Inc., Burlington, MA) was used to conduct the numerical simulations. The 2D computational domain is discretized into an unstructured mesh of triangular elements (Delaunay triangulation, Figure 3A). Since the flow is fully developed in the top channel, the particular choice of the top channel length [0 is immaterial and does not affect the results. The scaled top channel length iolil can be set to between 2 and 4, large enough for numerical convergence. The maximum mesh edge length was set to 0.025 in the bulk and 0.0025 at all corners and surface edges. The default convergence criteria in Comsol' s static FEM solver were employed. Though the number of elements can vary with the geometrical parameters, approximately 15,000 to 25,000 mesh elements and 1000 boundary elements were used in a single computation.

Example 1. Fabrication of a Microfluidic Device Comprising Cavities with 2 Surfaces

[00114] To fabricate the microfluidic device of the invention, e.g. having cavities with an upper and lower surface (Figure 1), silicon master molds were created for the top fluidic channel and for the bottom substrate comprising such cavities. The master mold for the 80- /m-high top fluidic channel was made using a negative photoresist (SU-8 2050, Microchem, MA).

[00115] The base substrates comprising cavities with varying surface depths (Table 1) were fabricated using a two-step photolithography method. The first layer of the microsurface patterns (surface depths hl= 20, 50, and 80 μηι) were fabricated using negative photoresists (SU- 8 2015, SU-8 2050). For the 20^m-deep pattern, SU-8 2015 was spin-coated at 2,000 rpm for 30 s, baked for 3 min at 95°C, and exposed to UV (140 mJ cm^"2) for 1 min. The 50- and 80-μηι- deep patterns were fabricated by spin-coating SU-8 2050 at 2,600 rpm and 1,700 rpm for 1 min, respectively. The second layer of the surface patterns was fabricated by spin coating SU-8 2050 photoresist on a silicon wafer patterned with the first surface patterns. The total depth of the first and second layers was 100 μηι and the depth ratios were h2lhl = 1/4, 1 and 4.

Table 1: Dimensions of double surfaces and inlet channels for fabricated surfaced microfluidic device.

[00116] Following photolithography, negative replicas of the top fluidic channel and the base substrate comprising cavities of the invention were molded in poly(dimethylsiloxane) (PDMS) (Sylgard 184 Silicon elastomer, Dow Corning, MI). The PDMS prepolymer mixed with silicone elastomer and curing agent (10:1 ratio) was poured on the silicon masters and cured at 70 °C for 2 h. The PDMS molds were subsequently peeled off the silicon masters. Inlet and outlet ports for cell loading and medium perfusion were created in the top fluidic channel using sharp punchers. The top fluidic channel and base substrate were aligned and irreversibly bonded using oxygen plasma (5 min at 30W, Harrick Scientific, NY).

[00117] SEM images of the cavity comprising an upper and lower surface are shown in Figure 2. Six geometries were fabricated involving two surface widths and three surface depth ratios (Figure 2C and Table 1)

Example 2. Numerical Simulation of Flow Pattern in Cavities with 2 Surfaces

[00118] A combined approach of dimensional analysis and numerical simulation were employed to rationalize experimental results and to assess flow and cell capture patterns for multi-surface cavities of a preselected geometry. Dimensional analysis is an efficient and systematic method of determining the dependence of flow patterns on the various geometrical and flow parameters. Computational fluid dynamics (CFD) is a powerful approach to solve many fluid dynamics problems [17-20]. Combined, these methods elucidate the dependence of the microcirculation pattern, shear stress, cell capture, and cell retention on the various geometric and flow parameters. Past reports have discussed the flow patterns on one surface within cavities [21-23]. However, these reports do not disclose or teach the flow patterns in cavities with more than one surfaces. [00119] For the purpose of modeling, it was assumed that the flow in the cavity of the invention is steady and two dimensional (2D). The flow is governed by the Navier-Stokes equations subject to appropriate boundary conditions (for details, see Materials and Methods discussed above). All spatial variables were scaled by the width [l of the upper surface and flow velocities were scaled by the average inlet velocity U=Q/(u 0li0). The flow in the cavities with two surfaces is then specified by the five dimensionless ratios Ιίθ/ίΐ, h2lhl, (Iil+li2)/[l, and [21 [1 (Table 2) and the Reynolds number Re=Q/( toOv) of the inlet channel (Table 3). In the experiments, the Reynolds number Re can range from 0.04 to 0.33, and hence the flow is laminar and close to the Stokes flow limit, where the scaled velocities, shear stresses, and pressure gradients are purely dependent on geometry and not on the flow rate [25].

[00120] The flow in cavities with two surfaces was simulated numerically (for details, see Materials and methods discussed above) over a broad range of geometries (Figure 3A) and flow intensities, i.e., Reynolds numbers Re. In the parameter regime of interest, five characteristic flow patterns arise based on the type of microcirculation present in the bulk, and are presented in Figure 3B: i) corner eddies on the upper and lower surfaces that are connected or ii) are connected separately on the upstream and downstream sides; iii) no circulation, iv) a double- eddy on the bottom surface, and v) a single eddy on the the bottom surface. Corner eddies exist in the four surface corners for all flow rates and geometries [28]. Simulations were run for a representative Reynolds number of Re=0.1; virtually identical patterns exist for Re between 0 and 1. At these small Reynolds numbers, the streamline patterns and velocity contours all have approximate fore-aft symmetry, since the flow is close to the Stokes flow limit, which depends purely on the fore-aft symmetric double surfaces. Configurations (iii-v) correspond to the fabricated geometries 1-3, respectively, listed in Tables 2 and 3, and differ only in surface depth ratio hZlhl. In general, narrow deep surfaces contain eddies (Figure 3B; Configurations iv and v), h2lhl=\£) while wide shallow surfaces do not (Figure 3B; Configuration iii, h2lhl=\l\). The exception occurs for high inlet channels and wide surfaces, i.e., large holil, where corner eddies on the upper surface drive recirculation in the lower surface (Figure 3B; Configuration i, ho/ [1=0.53, fi2/fil=0. l). Reducing holil causes the recirculation to disappear (Figure 3B;

Configuration ii, ho/ [1=0.50, h2lhl=0. X). Lastly, configurations (iii-v) in Figure 3B also include the dimensionless velocity contour plots, which show that the magnitude of the velocity decreases rapidly into the sheltered surfaces.

Bottom Shear Stress

[00121] Fluid shear stresses exerted on cells in microfluidic devices largely determine the cell capture pattern and retention. Cell adherence to channel surfaces decreases with increasing shear stress[29,30] and is also related to the flow behavior[23,31-35]. The cell capture patterns are given by the sign of the shear stress at the bottom boundaries of each surface. The bottom shear stress in the lower surface was demonstrated in Figure 3C, and the shear stress in the lower surface was calculated via

[00122] The shear stress on the inlet channel wall given by the standard result was non- dimensionalized for Poiseuille flow

where ho and <w0 are the channel inlet height and width, respectively. The bottom shear stress Ί is between 1% and 5% of that in the inlet channel, i , less than that for a single surface of the outer width, but generally greater than that for a single surface of the inner width

(Figure 3C). Also, 1_bot li;₀ is smaller for deeper and narrower surfaces, i.e., larger hZlhl and smaller [21 [1. This is rationalized by noting that the total viscous dissipation in the bottom surface scales as 2pve_ij ²w₂h₂l₂ ~ pvU ²w₂h₂ //₂ , where e_tj is the rate of strain tensor and U is the inlet flow speed [25]. Thus as h2 increases or [2 decreases, the viscous dissipation is increased in the bottom surface, the flow speed is reduced and the sheltering effect enhanced. Manbachi et al.[16] calculated the bottom shear stress on a single surface and have discussed that it decreases as the surface narrows. It was also previously reported that there is a linear dependence of bottom shear stress on inlet velocity U, and hence Reynolds number Re, since the flow is close to the Stokes regime. However, the report does not teach or suggest that the ratio of h2 to hi can affect the location of cell capture. In addition, cavities with one surface only do not allow a co-culture of cells at distinct locations.

Example 3. Cell Capture in the Cavities with 2 Surfaces

[00123] This study provides both theoretical rationale and experimental evidence for cell capture in one embodiment of a microfluidic device as disclosed herein. The experimental demonstration of cell capture and retention presented herein correlate with the simulated flow direction and bottom shear stress magnitude in the microfluidic device.

[00124] Experimental findings of cell capture in the four corners of the surfaces are presented in Figures 4A to 4C. The corresponding cell counts are presented in Figure 4D, sorted with respect to the cell-capture position/flow pattern type for each fabricated cavity geometry. Results for the two surface widths, [1 = 150 and 200μπι, are similar. Also, similar cell counts are observed on the upper and lower surfaces since the upper surfaces are (laterally) longer than the lower surfaces. Provided the cell diameter is small relative to the surface dimensions, cell capture and positioning are assessed solely based on the flow direction near the surface boundaries. The larger depth ratios h2lhl are associated with microcirculation in the bottom cavity, resulting in cells accumulating on the upstream surfaces. Smaller depth ratios h2lhl correspond to little or no microcirculation and hence cells accumulate on the downstream of the surfaces. Furthermore, cells were more dispersed in the deeper bottom surfaces as a result of the higher sheltering and weaker flows. To summarize, for cases where the cells are small relative to the surface dimensions, the microcirculation pattern is sufficient for predicting cell capture.

[00125] When surface dimensions become the same order as the cell diameters, cells are affected by both the interior flow in the bulk and the bottom shear stress. In particular, the sign of the shear stress at the surface boundary cannot be sufficient for determining the cell capture pattern. For example, the shallow lower surfaces in geometry 4 (Table 1 in Example 1) are 20 μηι deep, the same order as the cell diameter. Even though microcirculation exists in the thin lower surface cells accumulate downstream of the lower surface as if unaffected by the reversed flow.

Cell Retention in the Cavities with 2 Surfaces

[00126] Fluid flow through microfluidic devices delivers nutrients, growth factors, and reagents to the cells captured inside. However, exposing the cells to flow also leads to cell removal from the device. Therefore, surface designs that offer optimal cell retention at a given inlet flow velocity U are desirable. Figures 5A to 5B show the cell retention in the cavities with 2 surfaces after exposure to a given inlet flow velocity U. Cell retention is determined to decrease approximately linearly with increasing inlet flow velocity. Moreover, cell retention is generally less on upper surfaces than on lower surfaces, since cells on the upper surface are more easily removed than those on the sheltered lower surface. Cell retention was higher for narrower and deeper surfaces (larger (fil+fi2)/il and hZlhi), which are associated with higher viscous dissipation and thus lower flow speeds and shear stresses, as discussed herein.

[00127] The approximate linear dependence of cell retention on inlet velocity U and Reynolds number Re is rationalized by noting that cell mobility is proportional to shear stress [29] and, as discussed herein, the shear stress at a particular location varies linearly with U (or Re). Hence, at a particular location, cell retention should vary linearly with U (or Re).

[00128] Cell retention also varies with location on the surface, with low retention associated with exposed outer edges and higher retention with sheltered corners. Accordingly, the effects of position and flow velocity U (or Re) are confounded in the cell-retention measurements derived from cell counts over entire surfaces. These effects are partially separated in Figures 5C to 5D by plotting cell retention versus U, which reveal one or two stages of cell removal. Consistent with our previous discussion, in each stage the retention has a linear dependence on U (or Re). Exposed cells are rapidly removed first (steep slope), followed by the slow removal of sheltered cells (mild slope). The cell retention on lower surfaces (Figure 5C) is generally higher and more uniform, due to the relatively uniform sheltering, compared with that on upper surfaces (Figure 5D). The trends noted for Figures 5 A to 5B are also evident in Figures 5C to 5D: retention is higher for narrow and deep surfaces compared with wide and shallow surfaces.

[00129] Lastly, the maximum cell retentions in the single-surface cavity designs of Manbachi et al.[16] are plotted in Figure 5C for comparison, and are generally lower than that in the device of the invention for the same average flow speed U. Despite some of Manbachi et al.'s surfaces having aspect ratios up to 1.6, their shallow surface depths of 40um are of the same order as cell diameters. The cells could be affected by the interior flows, rather than simply the small bottom shear stresses, thereby reducing retention. The ratio of cell diameter to surface depth is crucial in cavity design when cell retention is important.

Example 4. Classification of Flow Pattern and Cell Capture

[00130] Experimental demonstration verified that cell capture are determined by the microcirculation pattern. Accordingly, it is important to relate the microcirculation pattern to the cavity geometry and inlet flow speed U (or Reynolds number Re). To this end, phase diagrams in Figure 6 of the microcirculation patterns were constructed in terms of the critical depth ratio hZlhl and scaled inlet height holil, for various scaled total depths (fi2+fil)/il, surface width ratios

[2/Cl, and Reynolds numbers Re. The phase boundaries are found by binary search based on the signs of the shear stress at the fore-aft edges between the upper and lower surfaces and at the middle of the lower surface. The binary search is stopped when the difference in parameter values separating two flow patterns is less than 0.001. The phase boundaries for the three Reynolds numbers Re=0, 0.1, 1 plotted in Figures 6A to 6C virtually coincide, demonstrating the insensitivity to U (and Re). The parameter regime is close to Stokes flow, where the dimensionless flow pattern is dependent only on the surface geometry, not the inlet velocity. Figures 6A to 6B show the phase diagrams corresponding to the fabricated double surfaced channel geometries 1-3 and 4-6 in Table 1 in Example 1, respectively. The phase boundaries accurately separate the different cell-capture patterns, except for the leftmost data square in Figure 6A in which the cell diameter is comparable to the surface depth, as discussed herein. Higher inlet aspect ratios holil and surface depth ratios h2lhl are associated with recirculation, which is consistent with the behavior noted for single surfaces since increasing h2lhl effectively increases the aspect ratio h2li2 of the lower surface. The phase boundary separating recirculation from no circulation approaches the corresponding cavity with one single surface limit as

L2/ LZ— >0, denoted by the asterisk (*). A second phase boundary separates coupled and decoupled eddies between the upper and lower surfaces (straight lines in Figures 6A to 6B). This phase boundary has no analog in the single surface, and thus does not have a corresponding single surface limit. The phase diagram for cavities with one single surfaces shows that higher inlet aspect ratios holil and surface aspect ratios hllil are associated with recirculation

(Figure 6C). The phase boundary accurately separates the flow and cell capture patterns observed by Manbachi et al.[16]. The dependence of the phase boundaries on the surface width ratio [21 [1 and scaled total depth (fi2+fil)/[l is shown in Figures 6D to 6E, respectively. Increasing the width ratio [21 [1 widens the lower surface, requiring a larger depth ratio h2lhl for

recirculation, thereby moving the phase boundary to the right in Figure 6D. Increasing the scaled total depth (fi2+fil)/[l increases the overall surface aspect ratio, reducing the depth ratio h2lhl required for recirculation, thereby moving the phase boundary to the left in Figure 6E.

Example 5. Effect of surface alignment on flow pattern and cell capture

[00131] Misalignment between the centers of the upper and lower surfaces (Figure 2B) breaks the symmetry of the surface geometry and the near symmetry of the flow. In the limit as the lower surface is shifted completely to one side, an edge and corner disappear along with the associated eddies. The effects of surface misalignment on the flow pattern are precisely assessed with numerical simulation. Streamline patterns for off-centered lower surfaces at Re=0.1 are shown in Figure 7A. Upstream shifts of the bottom surface cause the upper and lower corner eddies to couple on the upstream side of both surfaces, while not on the downstream side.

Downstream shifts of the lower surfaces cause eddy coupling on the downstream side while not on the upstream side. The effect of surface misalignment on the flow pattern phase boundaries at Re=0 is illustrated in Figures 7B and 7C for surface aspect ratios (fil+fi2)/[l = 2/3 and =1/2, respectively. Phase boundaries for upstream/downstream shifts of equal magnitude coincide and flow patterns are mirror images about surface center. As the lower surface is shifted upstream, the phase boundary that separates the coupled eddy phase from the separated eddy phase splits in two, creating new phases in which the upper and lower surface eddies are coupled on the upstream side, but separated on the other side. For one embodiment of the invention, e.g., a cavity with an upper and lower surface, Figures 7B and 7C demonstrate that misalignment of up to 1/8 of the upper surface width do not alter the characteristic flow patterns for designs that are relatively wide ((fil+fi2)/ [1=1/2) or have deep lower surfaces (fi2/fil>l). However, the top-bottom coupled eddy flows in narrow double surfaces with a shallow lower surface can be affected by small misalignment. For example, shifting the bottom surface in geometry 4 (Table 2 in

Example 2, (hl+fi2)l [1=2/3 and h2lhl=\l\) has the effect shown in Figure 7A, which persists with left/right shifts as low as 1/16 of the top surface width. As disclosed herein, cell capture occurred downstream in geometry 4 despite the recirculation present in the lower surface; a shift of the bottom surface can be partially responsible. Example 6. Cell Capture in Other Geometries

[00132] When fluid flows past a sharp corner of surfaces within a cavity, cells can be effectively captured and immobilized if the corner affords sufficient shear protection. Without wishing to be bound by theory, a myriad of geometries can be employed to create various cell- capture patterns, such as multi-surface cavities of rectangular, triangular, trapezoidal, or other cross sections (see Reference [16] and references therein). Computational flow simulations analogous to those demonstrated herein can help design the cell-capture patterns and the degree of shear protection. The particular choice of surface geometry depends not only on the concomitant cell-capture pattern but can also depend on the fabrication viability, mass-transport properties, and the area afforded for cell adhesion. Such features are important for the design of biosensors in order to deliver nutrients and diagnostic chemicals to immobilized cells.

[00133] Cell capture and retention within cavities comprising one or more surfaces at different depths therein in a microfluidic device have been demonstrated through a combined theoretical and experimental approach. Bi-modal cell-capture patterns that result in the upper and lower surfaces have been demonstrated. Further, the links of the streamline pattern and shear stress direction to cell capture have been demonstrated. The phase diagrams of the

microcirculation pattern have been constructed as a function of the four dimensionless ratios that specify the cavity geometry with an upper and lower surface. Accordingly, given a particular design of cavities with 2 surfaces, the microcirculation pattern can be found from the phase diagrams, allowing accurate estimates of the cell capture. The degree of cell retention on the upper and lower surfaces and its dependence on surface geometry and its linear dependence on inlet flow speed have also been qualified. The larger depth of surfaces with respect to cell diameter also increases cell retention compared with that previously reported for cavities with one single surface. Microfluidic devices of the invention, comprising cavities with more than one surfaces, provide controlled cell capture while affording shear protection and high cell retention, and are thus ideally suited for cell-based biosensors and high-throughput drug screening.

REFERENCES

All references cited herein, in the specification and Examples are incorporated in their entirety by reference.

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[00134] It is understood that the foregoing detailed description and examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

What is claimed is:

1 . A microfluidic device comprising:

2. The microfluidic device of claim 1, further comprising a top substrate mounted on the top surface of the base substrate, the top substrate defining at least one channel extending over the base substrate, wherein the at least one channel is in communication with the cavity on the base substrate and the at least one channel has a predefined height ho.

3. The microfluidic device of claim 1, wherein the first surface and the second surface are concentric.

4. The microfluidic device of claim 1, wherein the first surface has a center and the second surface has a center and the center of the first surface is not aligned with the center of the second surface.

5. The microfluidic device of claim 1, wherein the first surface or the second surface is asymmetric with respect to an axis transverse to the at least one channel.

6. The microfluidic device of claim 1, wherein at least one of the first surface and the

second surface have an irregular shape.

7. The microfluidic device of claim 1, wherein at least one of the first surface and the

second surface have a regular shape.

8. The microfluidic device of claim 7, wherein the regular shape is selected from a group comprising a circle, a square, a rectangle, a triangle, a trapezoid, and a polygon.

9. The microfluidic device of any of claims 1 to 8, wherein the first surface has a

predefined width il and the second surface has a predefined width (2, wherein (l, [2 are determined as a function of ho, hi, h2, a desired number of cells on each of the first and the second surfaces, corner eddies, a shear stress profile in the cavity, and properties of fluid.

10. The microfluidic device of claim 9, wherein the desired number of cells on each of the first and the second surfaces is determined as a function of binding affinity of cells to the base substrate, cell retention time, flow rate of a fluid passing through the channel in the top substrate and area of the first and the second surfaces.

11. The microfluidic device of claims 2 and 9, wherein ho is determined as a function of il and properties of fluid.

12. The microfluidic device of claims 9 and 11, wherein the properties of fluid comprise a flow rate of a fluid passing through the channel in the top substrate, and a density and viscosity of the fluid.

13. The microfluidic device of any of claims 1 to 12, wherein the ratio of ho to tl is less

than 1.

14. The microfluidic device of any of claims 1 to 12, wherein the ratio of ho to tl is in the range of about 0.2 to about 0.7.

15. The microfluidic device of any of claims 1 to 12, wherein tl is greater than t2.

16. The microfluidic device of any of claims 1 to 12, wherein the ratio of tl to t2 is about 2.

17. The microfluidic device of any of claims 1 to 12, wherein hi and h2 are each

independently greater than a cell diameter.

18. The microfluidic device of any of claims 1 to 12, wherein hi is at least about ΙΟμηι.

19. The microfluidic device of any of claims 1 to 12, wherein hi is at least about 20μηι.

20. The microfluidic device of any of claims 1 to 12, wherein hi is at least about 30μηι.

21. The microfluidic device of any of claims 1 to 12, wherein h2 is at least about ΙΟμπι.

22. The microfluidic device of any of claims 1 to 12, wherein h2 is at least about 20μπι.

23. The microfluidic device of any of claims 1 to 12, wherein h2 is at least about 30μπι.

24. The microfluidic device of any of claims 1 to 12, wherein the ratio of h2 to hi is any

value greater than 1.

25. The microfluidic device of any of claims 1 to 12, wherein the ratio of h2 to hi is equal to about 1.

26. The microfluidic device of any of claims 1 to 12, wherein the ratio of h2 to hi is any

value less than 1.

27. A method of making a microfluidic device, the method comprising:

providing a base substrate having a top surface;

forming the first surface at a predefined distance hi below the top surface;

28. The method of claim 27, wherein the first surface and the second surface are concentric.

29. The method of claim 27, wherein the first surface and the second surface are asymmetric.

30. The method of claim 27, wherein the first surface or the second surface is asymmetric.

31. The method of claim 27, wherein at least one of the first surface and the second surface have an irregular shape.

32. The method of claim 27, wherein at least one of the first surface and the second surface have a regular shape.

33. The method of claim 32, wherein the regular shape is selected from a group comprising a circle, a square, a rectangle, a triangle, a trapezoid, a polygon and the like.

34. The method of any of claims 27 to 33, wherein the first surface has a predefined width il and the second surface has a predefined width (2, wherein il and [2 are determined as a function of ho, hi, h2, a desired number of cells on each of the first and the second surfaces, corner eddies, a shear stress profile in the cavity, and properties of fluid.

35. The method of claim 34, wherein the desired number of cells on each of the first and second surfaces is determined as a function of binding affinity of cells to the base substrate, cell retention time and flow rate of the fluid passing through the channel in the top substrate.

36. The method of claims 27 and 34, wherein ho is determined as a function of il and

properties of fluid.

37. The method of claims 34 and 36, wherein the properties of the fluid comprise a flow rate of the fluid passing through the channel in the top substrate, and a density and viscosity of the fluid.

38. The method of any of claims 27 to 37, wherein the ratio of ho to il is less than 1.

39. The method of any of claims 27 to 37, wherein the ratio of ho to il is in the range of about 0.2 to about 0.7.

40. The method of any of claims 27 to 37, wherein il is greater than [2.

41. The method of any of claims 27 to 37, wherein the ratio of il to [2 is about 2.

42. The method of any of claims 27 to 37, wherein hi and h2 are each independently greater than about a cell diameter.

43. The method of any of claims 27 to 37, wherein hi is at least about ΙΟμηι.

44. The method of any of claims 27 to 37, wherein hi is at least about 20μπι.

45. The method of any of claims 27 to 37, wherein hi is at least about 30μπι.

46. The method of any of claims 27 to 37, wherein h2 is at least about ΙΟμπι.

47. The method of any of claims 27 to 37, wherein h2 is at least about 20μπι.

48. The method of any of claims 27 to 37, wherein h2 is at least about 30μπι.

49. The method of any of claims 27 to 37, wherein the ratio of h2 to hi is greater than 1.

50. The method of any of claims 27 to 37, wherein the ratio of h2 to hi is equal to about 1.

51. The method of any of claims 27 to 37, wherein the ratio of h2 to hi is less than 1.

52. A method for capturing at least one cell in at least one cavity of a microfluidic device, the method comprising:

providing a microfluidic device of claim 2; and

53. The method of claim 52, wherein the first cell-containing fluid flows at a flow rate Ql through the at least one channel.

54. The method of claims 52 and 53, wherein the first cell-containing fluid comprises at least a first cell population.

55. The method of any of claims 52 to 54, wherein the first cell-containing fluid further comprises at least a second cell population.

56. The method of claim 52, wherein at least one cell is captured on the first surface of the cavity.

57. The method of claim 52, wherein at least one cell is captured on the second surface of the cavity.

58. The method of claim 52, wherein at least one cell is captured on an upstream portion of the first surface of the cavity.

59. The method of claim 52, wherein at least one cell is captured on an upstream portion of the second surface of the cavity.

60. The method of claim 52, wherein at least one cell is captured on a downstream portion of the first surface of the cavity.

61. The method of claim 52, wherein at least one cell is captured on a downstream portion of the second surface of the cavity.

62. The method of any of claims 56 to 61, wherein a position of the cells captured on the first surface or the second surface can be controlled as a function of a ratio of h2 to hi.

63. The method of claim 62, wherein the ratio of h2 to hi is greater than 1.

64. The method of claim 62, wherein the ratio of h2 to hi is equal to about 1.

65. The method of claim 62, wherein the ratio of h2 to hi is smaller than 1.

66. The method of claim 52, further comprising flowing at least a second fluid at a flow rate Q2 through a second inlet of at least a second channel in the top substrate of the microfluidic device.

67. The method of claim 66, wherein the second fluid is same as the first cell-containing fluid.

68. The method of claim 66, wherein the second fluid is cell-free.

69. The method of claim 66, wherein the second fluid contains at least a third cell

population.

70. The method of claim 55 wherein the first and second cell populations are prokaryotic cells.

71. The method of claim 69, wherein the third cell population is prokaryotic cells.

72. The method of claim 55 wherein the first and second cell populations are eukaryotic cells.

73. The method of claim 69, wherein the third cell population is eukaryotic cells.

74. The method of any of claims 72 to 73, wherein the eukaryotic cells are mammalian cells.

75. The method of any of claims 52 to 74, wherein Ql is equal to Q2.

76. The method of any of claims 52 to 74, wherein Ql is greater than Q2.

77. The method of any of claims 52 to 74, wherein Ql is smaller than Q2.

78. The method of any of claims 52 to 77, further comprising

79. The method of any of claims 52 to 78, wherein the flow is laminar flow.

providing a microfluidic device of claim 2; and

81. The method of claim 80, wherein the first fluid flows at a flow rate Ql through the at least a one channel.

82. The method of claim 80, further comprising flowing at least a second fluid at a flow rate Q2 through a second inlet of at least a second channel in the top substrate of the microfluidic device.

83. The method of claim 80, wherein the first fluid comprises at least a first cell population.

84. The method of claim 80, wherein the first fluid further comprises two or more cell

populations.

85. The method of claim 82, wherein the second fluid is same as the first fluid.

86. The method of claim 82, wherein the second fluid is cell-free.

87. The method of claim 82, wherein the second fluid comprises at least a third cell type.

88. The method of claims 80 and 87, wherein the first, second and third cell types are

prokaryotic cells.

89. The method of claims 80 and 87, wherein the first, second and third cell types are

eukaryotic cells.

90. The method of claim 89, wherein the eukaryotic cells are mammalian cells.

91. The method of any of claims 80 to 90, wherein at least one cell is captured on a first surface of the cavity.

92. The method of any of claims 80 to 90, wherein at least one cell is captured on a second surface of the cavity.

93. The method of any of claims 80 to 90, wherein at least one cell is captured on an

upstream portion of the first surface.

94. The method of any of claims 80 to 90, wherein at least one cell is captured on an

upstream portion of the second surface.

95. The method of any of claims 80 to 90, wherein at least one cell is captured on a

downstream portion of the first surface.

96. The method of any of claims 80 to 90, wherein at least one cell is captured on a

downstream portion of the second surface.

97. The method of any of claims 80 to 96, wherein a position of the cells captured on the first surface or the second surface can be controlled as a function of a ratio of h2 to hi.

98. The method of claim 97, wherein the ratio of h2 to hi is greater than 1.

99. The method of claim 97, wherein the ratio of h2 to hi is equal to about 1.

100. The method of claim 97, wherein the ratio of h2 to hi is smaller than 1.

101. The method of claim 82, wherein the second channel is orthogonal to the at least one channel.

102. The method of any of claims 80 to 101, wherein Ql is equal to Q2.

103. The method of any of claims 80 to 101, wherein Ql is greater than Q2.

104. The method of any of claims 80 to 101, wherein Ql is smaller than Q2.

105. The method of any of claims 80 to 104, further comprising

106. The method of any of claims 80 to 105, wherein the flow is laminar flow.