Examples Of Advanced Microfluidic Cellular Bioassays

Inexpensive rapid microfabrication techniques combined with knowledge of microscale phenomena (e.g. laminar flows and dominance of surface tension) allow development of new advanced microfluidic cellular bioassays. Microfluidic devices for these applications typically have cross-sectional capillary dimensions of 10-500 pm. This size scale is similar to those of biological cells, so that these devices are well suited to the transport, manipulation, and chemical or biochemical treatment of single cells or small number of cells [48]. This section describes several specific systems that take advantage of microfluidics to create controlled microenvironments to study subcellular phenomena, to sort cells by function such as motility, and to analyze cells. These advanced systems allow for the microscale control of interfaces that have not been exploited in traditional systems.

4.4.1. Patterning with Individual Microfluidic Channels

The most straightforward application of microfluidics is the use of different channels to pattern different cells or molecules in different regions on the microscale. The small size of microfluidic channels and the ability to fabricate multiple channels in parallel provides a platform for simultaneously patterning different biomolecules onto a surface.

Delamarche et al. used microfluidic networks for the spatially controlled deposition of biomolecules onto various substrates. The networks are constructed using a PDMS mold

FIGURE 4.2. Phase-contrast optical micrographs of hepatocytes patterned onto various polymers (A: Polystyrene Petri dish, B: PDMS sheet, C: Polycarbonate Petri dish, and D: Poly(methyl methacrylate) disk) 12-24 hours after seeding. Hepatocytes adhere only to regions of collagen (A and B) or fibronectin (C and D). Scale bar is 50 |im. [23] (Reprinted with permission from Folch 1998. Copyright 1998 American Chemical Society)

FIGURE 4.2. Phase-contrast optical micrographs of hepatocytes patterned onto various polymers (A: Polystyrene Petri dish, B: PDMS sheet, C: Polycarbonate Petri dish, and D: Poly(methyl methacrylate) disk) 12-24 hours after seeding. Hepatocytes adhere only to regions of collagen (A and B) or fibronectin (C and D). Scale bar is 50 |im. [23] (Reprinted with permission from Folch 1998. Copyright 1998 American Chemical Society)

sealed to a surface to form a conduit. The capillaries are filled with solutions containing the biomolecules of interest, allowed to deposit onto the surface, and the PDMS later peeled off. Using this type of system, immunoglobulins were patterned onto substrates with submicron resolution and used in enzyme-linked immunosorbent assay (ELISA)-type assays [16].

Microfluidics allows manipulation of cellular co-cultures in ways not possible with macroscopic techniques. Toner et al. have selectively delivered different cell suspensions in laminar flows to tissue culture substrates. Another technique uses microchannels saturated with protein solutions that adsorbs onto the surface exposed to the microflow. After microchannels are removed, only the proteins remain and cells attach selectively to these regions. Micropatterned cocultures have been created using patterns of collagen or fibronectin (Figure 4.2) [22].

Whitesides et al. have used three-dimensional (3D) microfluidic systems to pattern proteins and cells onto a surface. 3D microfluidic stamps are fabricated using a two-step photolithographic process and sealing two PDMS slabs together. This PDMS structure is then used as a conduit to deliver proteins (bovine serum albumin and fibronectin fluores-cently labeled) and cells (bovine capillary endothelial cells (BCEs) and human bladder cancer cells (ECVs) shown in Figure 4.3). This technique allows for the creation of biologically relevant patterns on surfaces with only one additional step of pattern fabrication, alignment, and sealing [10].

Jeon et al. have developed a microfabricated neuronal culture device. The system is created from PDMS and consists of two compartments separated by a physical barrier

FIGURE 4.3. A 3D PDMS stamp shown in (A) is used to deposit two cell types onto a tissue culture dish in a concentric square pattern. Fluorescence (B) and phase-contrast (C and D) pictures show ECVs labeled in green CMFDA and BCEs labeled in a red Dil-conjugated acetylated low density lipoprotein. The cells are cultured with the stamp in place for 24 hours to grow and spread to confluency. The pictures are taken immediately after removing the stamp. (D) is an expanded view of the lower right corner in (C) [10]. (Reprinted with permission from Chiu, Jeon et al. 2000, Copyright 2000 National Academy of Sciences, USA).

FIGURE 4.3. A 3D PDMS stamp shown in (A) is used to deposit two cell types onto a tissue culture dish in a concentric square pattern. Fluorescence (B) and phase-contrast (C and D) pictures show ECVs labeled in green CMFDA and BCEs labeled in a red Dil-conjugated acetylated low density lipoprotein. The cells are cultured with the stamp in place for 24 hours to grow and spread to confluency. The pictures are taken immediately after removing the stamp. (D) is an expanded view of the lower right corner in (C) [10]. (Reprinted with permission from Chiu, Jeon et al. 2000, Copyright 2000 National Academy of Sciences, USA).

with embedded micron-sized grooves. Neuronal cells are cultured into one compartment and after several days, neurites extend through the grooves and into the second compartment (Figure 4.4). They are also able to micropattern neurites on the surface through microcontact printing to direct neuronal attachment and orientation of the neuronal outgrowth [84].

4.4.2. Multiple Laminar Streams

In the previous section, each microfluidic channel transported one type of molecule or cell. The low Reynolds number flow characteristics in microfluidic channels, however, permits two or more streams of cells and biomolecules to flow next to each other inside of a single microfluidic channel without turbulent mixing. The only mixing at the interface between different streams takes place through diffusion. Researchers have exploited laminar flow in microfluidic networks to pattern proteins, cells, and planar lipid bilayers on substrates with micrometer-scaled resolution [58]. Multiple laminar streams are also

FIGURE 4.4. Neuronal cell culture inside microfabricated device. Calcein AM and Texas Red dextran are added to the neuritic chamber 1 hour before taking the pictures. (A) Phase micrograph after 4 days in culture of neurons extending to neuritic chamber (on the right). (B) Epifluorescence micrograph of same region with cells stained in green calcein AM, and the neuritic chamber in red dextran [84]. (Reprinted with permission from Taylor, Rhee et al. 2003. Copyright 2003 American Chemical Society)

FIGURE 4.4. Neuronal cell culture inside microfabricated device. Calcein AM and Texas Red dextran are added to the neuritic chamber 1 hour before taking the pictures. (A) Phase micrograph after 4 days in culture of neurons extending to neuritic chamber (on the right). (B) Epifluorescence micrograph of same region with cells stained in green calcein AM, and the neuritic chamber in red dextran [84]. (Reprinted with permission from Taylor, Rhee et al. 2003. Copyright 2003 American Chemical Society)

useful in generating patterns or interfaces and gradients composed of (1) different adhesive regions, (2) different cell types, and (3) different solutions [79].

Multiple laminar streams can also generate and maintain gradients of chemicals. Cells naturally migrate in gradients of soluble molecules called chemoattractants in a process known as chemotaxis. In order to further examine this dynamic behavior, Jeon, Toner, Whitesides et al. have developed a technology that generates a stable, soluble chemoat-tractant gradient using a device fabricated by soft lithography. The device consists of a network of microfluidic channels with a gradient-generating portion and an observation

FIGURE 4.5. Schematic diagram of the microfluidic gradient generator. (A) Top view of device with gradient generating and observation regions. (B) 3D view of observation region where cells are exposed to chemoattractant gradients. (C) Micrograph of cells at the beginning of the experiment (0 min, left panel) deposited at the bottom of the field of view, and at the end of the experiment (90 min, right panel). Migration is subjected to a linear increase in IL-8 (0-50 ng/ml). Bar, 200 |im [40]. (Reproduced by permission from Jeon, Baskaran et al. 2002. Copyright 2002 Nature Publishing Group www.nature.com)

FIGURE 4.5. Schematic diagram of the microfluidic gradient generator. (A) Top view of device with gradient generating and observation regions. (B) 3D view of observation region where cells are exposed to chemoattractant gradients. (C) Micrograph of cells at the beginning of the experiment (0 min, left panel) deposited at the bottom of the field of view, and at the end of the experiment (90 min, right panel). Migration is subjected to a linear increase in IL-8 (0-50 ng/ml). Bar, 200 |im [40]. (Reproduced by permission from Jeon, Baskaran et al. 2002. Copyright 2002 Nature Publishing Group www.nature.com)

portion. (Figure 4.5A) The gradient-generating portion consists of a pyramidal branched array of channels that split, combine, and mix fluid streams. The channels recombine in the main channel to form a well-defined concentration gradient that spanned the width of the channel. (Figure 4.5B).

A gradient of interleukin-8 (IL-8) was formed in the device and the migration of neu-trophils was observed. Figure 4.5C shows the results of one experiment where cells are initially positioned at the side of lower IL-8 concentration and subjected to a linear increase in IL-8 (1-50 ng/ml). The cells move towards the higher concentration. The ability to generate and maintain stable linear gradients allowed straightforward determination of chemotaxis coefficients demonstrating the power of microfluidics for quantitative cell biology. Comparison of cell migration in stable cliff-shaped and hill-shaped gradient profiles also allowed observation of complex migratory behaviors of the neutrophils previously unappreciated [40]. Whitesides et al. have also created substrate-bound gradients of proteins using laminar flows in microchannels. Linear gradients are created through streams that flow through long serpentine channels where mixing occurs. All streams converge into one channel where the gradient is established. Gradients of laminin and bovine serum albumin are demonstrated and neuronal axon orientation studied [18].

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