Chemical Analysis of Single Mammalian Cells with Microfluidics
C
ells are the fundamental building blocks of life. All basic physiological functions of multicellular organisms reside ultimately in the cell. The misregulation of cellular physiology results in disease at the organism level. Thus, comprehending cell physiology is key to understanding and curing diseases. Many physiological processes can be studied using populations of cells. Others occur either on short timescales (e.g., kinase signaling cascades) or nonsynchronously (e.g., response to an external chemical gradient), so that taking a population average will not lead to an understanding of how the cellular chemistry occurs. These types of processes require single-cell analysis, and thus discretion must be exercised when deciding under which circumstances bulk versus singlecell analyses are more appropriate (1). In addition, many diseases like cancer start with a sinStrategies for culturing, sorting, gle cell; therefore, if one would like to find the trapping, and lysing cells and rare mutations in populations of cells that herald the inception of a disease, then cells must be separating their contents on chips. examined individually. Probing behavior at the single-cell level, however, is a very challenging task, primarily because of the small sample volume, the low abundance of material, and the fragile nature of the cell itself. Analyzing the contents of a single cell requires sensitive detection techniques and handling procedures that do not stress or damage it. Additionally, no proper blank exists that can be used, so truly quantitative studies are difficult. Intense interest in single-cell physiology, however, is driving the analytical and biomedical engineering fields to improve the technology for examining cells. One of the most popular and promising areas is lab-on-a-chip devices to manipulate and analyze single cells. Since Jorgenson’s groundbreaking work in 1989, CE has been used to examine the contents of single cells (2–4). However, many procedures for cell injection and lysis are time-consuming, and accuracy and reproducibility rely heavily on the skill of the researcher. Furthermore, the limited number of architectures provided by microbore tubing and its relatively large volume restrict the types of processes that can be investigated. Conversely, microfluidics, or lab-on-a-chip technology, offers a versatile format in which biological cells can be analyzed. These miniaturized devices provide several analytical and operational advantages over conventional macroscale systems (5, 6). Microfluidic architectures provide precise spatial control over reagents and samples, are capable of Alexander K. Price fast analysis times, can be automated, and can precisely manipulate picoliter Christopher T. Culbertson volumes of material without dilution. In addition, microfluidic systems are Kansas State University amenable to many different detection schemes, can be manufactured from many different materials at relatively low cost, allow flexibility of design, and provide the capability of integrating a series of multiple tasks (sample preparation, mixing, separation, etc.) in both serial and parallel schemes. Portable versions of these systems consume little power and only small quantities of reagents. These qualities allow the researcher to tailor a microfluidic system exactly to the experimental objective. Regardless of the chemistry being performed, the high-throughput capability and increased sensitivity of these devices, as well as the ability to construct cell-friendly microenvironments, have drawn increasing numbers of bioanalytical chemists to microfluidics. To perform a complete chemical analysis of cells on microfluidic devices, a number of cell handling and transport operations have to be integrated © 2007 AMERICAN CHEMICAL SOCIETY
A P R I L 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
2615
fluid flow during medium perfusion and allow for the facile removal of air bubbles. Cells can also be patterned on modified glass and native polystyrene (PS) substrates and incorporated into microfluidic devices (8). This technique requires that the substrate be exposed to an oxygen plasma that will etch away a layer of exposed polyOn-chip cell culture The first step in realizing the ideal total-cell analysis system is to L-lysine (cell-adhesive) or oxidize PS (PS is not cell-adhesive, incorporate a culture chamber so that as cells proliferate they can whereas oxidized PS is). The raised areas of a PDMS mold placed be sequestered in a different part of the microchip to undergo in contact with the substrate prior to etching define the areas of chemical analysis. Developing long-term cell-culture strategies cell adhesion. The plasma also sterilizes the device and promotes irreversible bonding of the substrate with a PDMS layer. When seeding is done, cells will occupy only the regions of the device that are defined for them. Devices fabricated with this method have demonstrated culture for up to 6 Cell culture days. This technology could be used in Cell sorting a more comprehensive device to keep cells from proliferating into analysis re7.5 days gions without the use of valves. The sophistication and flexibility of 130 µm – cell-culturing systems on microfluidic devices can be improved by creating a Reagent loading fluidic manifold that incorporates heating and feeding elements in place of an incubator. In these devices, a highthroughput cell-culture array can be fabricated out of PDMS by a two-step lithography process (9). The array is Lysis composed of large, circular chambers Separation intersection where the cells are cultured and two types of channels that define fluid flow. + One set of channels (~100 40 µm) serves to load cells and deliver reagents; a collection of smaller channels (2 2 FIGURE 1. Schematic of one idealized total-cell analysis device showing the various functions. µm) continuously perfuses the cells (Images adapted or adapted with permission from [clockwise starting at top left] Refs. 9, 14, 19, and 26.) with fresh medium, providing rapid and that are compatible with the rest of the on-chip procedures is dif- uniform fluid distribution. An indium tin oxide heater is used to ficult. Cells are particularly sensitive to their environments, and keep the temperature constant. Seeded HeLa cells reach confluan accurate assay will depend upon the success of the culture ency after 8 days without the use of an external incubator. This concept can be taken a step further by integrating a technique. Before the design process even begins, three issues require a good deal of forethought: providing proper growth con- PDMS channel manifold with a novel valving, mixing, and ditions (i.e., adding fresh media; removing wastes; preventing air pumping methodology based on a piezoelectric Braille display bubbles; maintaining proper temperature, pH, CO2, and hu- (10). When actuated, the Braille pins move upward, pressing a midity levels; etc.); harvesting cells; and transporting them from PDMS membrane into the channel and pinching it shut. With this format, specific regions of the microchip where cell seeding the culture chamber to the different microchip architectures. These issues are being tackled by various groups, and several will occur can be delineated and portions of this cell-culture very interesting designs demonstrating cell culture on microflu- channel can be split into several independent perfusion circuits idic devices have been reported recently. Most of these devices (Figure 2). These architectures could prove very beneficial for use the elastomeric polymer PDMS as a substrate capable of single-cell analysis because segments of the same cell population long-term cell culture (7 ). PDMS is nontoxic, gas-permeable, could be exposed to different chemical reagents, harvested, and and optically transparent—qualities that make it suitable for this analyzed sequentially. Moreover, the inclusion of a transparent application. The proliferation of mammalian cells in PDMS cul- heating unit and modification of cell medium allow for the culture microchambers can be observed with microscopy over the ture of C2C12 myoblasts and MC3T3-E1 osteoblasts for up to course of 8–10 days. Furthermore, cells grow to confluency in 3 weeks in nonsterile environments without the use of an microchambers with optimal geometries that provide uniform incubator. with high-efficiency separations and detection. Figure 1 shows a generalized schematic of the types of functional elements that may be needed on such a device. This article will discuss each of these components.
2616
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 , 2 0 0 7
In addition to the use of innovative geometries to enhance ~10 –15 µm, depending on the specific cell line. The walls of the on-chip cell culture, some work has explored novel substrate ma- channels may need to be treated with reagents to minimize cell terials. For example, normal murine mammary epithelial cells can adhesion and clumping. In addition, the means by which cells are be grown on enzyme-cross-linked gelatin substrates (11). These transported can affect the natural state of the cells. The large gelatin microchips can be made rather easily by casting a mixture electric potentials applied in microfluidic devices that transport or sort living cells via electrokinetic of gelatin and microbial transglutaflow and dielectrophoresis disrupt the minase solutions onto a PDMS mold (a) cell membrane; alter cell chemistry; and allowing it to gel. Although and, in some cases, induce apoptosis. PDMS is conducive to cell growth, Therefore, these transport mechagelatin-based microchips may pronisms may be of limited use. A final vide environments that more accuconsideration is that specific sorting rately mimic in vivo conditions bemethodologies may perform better cause the substrate closely resembles when the cells are present in high or the collagen-rich extracellular matrix low concentrations and should reflect to which the cell naturally adheres. the purpose of that particular device. Preliminary results show that cells Microfluorescence-activated cell adopt a different morphology on sorters (µFACSs) operate on the basic gelatin chips than on simple gelatin- (b) principle that a fluorescence signal treated plastic and actually proliferatfrom a stained cell triggers the operaed in a 3D manner, including into tion of valves to control flow switching the gelatin. (13). These devices can separate fluoIn another recent study, rerescent beads in a complex cell sample searchers immobilized HepG2 cells at a rate of ~12,000 cells/s, and 100in a peptide hydrogel formed in a Cell fold enrichments of bead concentraPDMS device (12). The cells are location tion can be obtained. The high sorting mixed with the Puramatrix sol-phase rates of µFACSs rival those of their hydrogel and focused down the cenbenchtop counterparts, but exact ter of a large channel. On contact Pump placement of the channel above the with cell media delivered from the microscope objective is required to ensheathing channels, the hydrogel sure proper time coordination. Moretransitions to the gel phase. Akin to over, such high sorting rates may jeopcells grown in gelatin microchips, ardize cell health, because the requisite cells encapsulated in this 3D miFlow direction high flow rates can produce significant croenvironment also exhibited a diflevels of shear stress. Although such ferent morphology than those grown high-throughput capabilities do not on planar substrates. However, the FIGURE 2. (a) Microchip showing an array of miappear to be desired for a totally intelargest impediment to incorporating crochannels for use with an integrated fluid control grated device performing single-cell cell-encapsulating sol–gels on a cell system. Sections of the circuit containing seeded cells analysis, this system demonstrates that analysis device is the current inability can be isolated from one another and independently additional components, such as culto free the cells so they can be trans- perfused. (b) Expanded view of the microchannel. ture chambers and optics, can be ported to other areas of the mi- (Adapted with permission from Ref. 10.) added to enhance functionality. crochip. Although it is too early to A more cell-friendly sorting mechanism (though it does not suggest that assays of cells grown in biomimetic environments may be more biologically accurate than assays performed on cells have the same performance) is to increase the sample purity by grown on other substrates, the possibility exists and warrants fur- redirecting unwanted cells from one flow stream into an adjacent stream (14). This can accomplished by applying a small voltage ther exploration. (5–15 V) between two microfabricated agarose gel electrodes positioned on both sides of the sorting junction. The electrokiCell sorting After cultured cells are harvested or cells from a complex biolog- netic effects transfer unwanted cells over to a waste channel while ical sample are introduced, being able to sort the overall cell pop- the cells of interest are shielded from any electric fields (Figure 1, ulation into subpopulations is advantageous, especially when “cell sorting”). Cells are recognized on an individual basis via only one group of cells is of interest. This requires that cells be high-resolution fluorescence imaging of the distribution of intratransported from one region of the microchip to another, for cellular contents. This process could be automated to increase which many other factors must be considered. For example, the ease of operation and reduce operator error. Elegant methodologies also exist for sorting cells on a mimicrofluidic channels must be large enough to accommodate the passage of mammalian cells, which usually have diameters of crofluidic device in an automated fashion without excessive exA P R I L 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
2617
(a) –P –P –P
Pulsed (b) compound flow –P –P Constant –P buffer flow
Pulsed compound flow Constant buffer flow
is 80%) regardless of whether the target-cell concentration in the sample is 50% or 1%. Although the laser beam operates at a high power, the heat generated by such a short pulse has a negligible adverse effect on the cell.
Cell trapping and reagent delivery 50 µm
Buffer (Qmain)
Compound (Pside)
(c)
(e)
(d)
33 ms
66 ms (f)
297 ms
0 ms
20 µm
FIGURE 3. (a) Schematic of a microfluidic network designed to capture individual cells and stimulate them with a short burst of reagent. (b) Expanded scanning electron microscope image of the described channel geometry. (c–f) Images of a pulse of reagent stimulating three trapped cells in
