Article pubs.acs.org/Langmuir
Cellular Microfabrication: Observing Intercellular Interactions Using Lithographically-Defined DNA Capture Sequences Hiroaki Onoe,†,‡ Sonny C. Hsiao,† Erik S. Douglas,§ Zev J. Gartner,†,∥ Carolyn R. Bertozzi,†,⊥,# Matthew B. Francis,†,# and Richard A. Mathies*,†,§ †
Department of Chemistry, University of California, Berkeley, California 94720, United States UCSF/UC Berkeley Joint Graduate Group in Bioengineering, University of California, Berkeley, California 94720, United States ⊥ Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, California 94720, United States # Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, United States §
S Supporting Information *
ABSTRACT: Previous reports have shown that synthetic DNA strands can be attached to the plasma membrane of living cells to equip them with artificial adhesion “receptors” that bind to complementary strands extending from material surfaces. This approach is compatible with a wide range of cell types, offers excellent capture efficiency, and can potentially be used to create complex multicellular arrangements through the use of multiple capture sequences. In this work, we apply an aluminum “lift off” lithography method to allow the efficient generation of complex patterns comprising different DNA sequences. The resulting surfaces are then demonstrated to be able to capture up to three distinct types of living cells in specific locations. The utility of this approach is demonstrated through the observation of patterned cells as they communicate by diffusion-based paracrine signaling. It is anticipated that the ability of this technique to create virtually any type of 2D heterogeneous cell pattern should prove highly useful for the examination of key questions in cell signaling, including stem cell differentiation and cancer metastasis.
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INTRODUCTION The micropatterning of living cells on a chip surface is an increasingly popular technique in cell biology, as it can enable single cell experiments in microfluidic devices, facilitate the observation of cell communication and differentiation, guide the generation of artificial tissues, and provide new types of cellbased chemical sensors.1,2 Several techniques have been used to direct the microscale arrangement of cells, such as the chemical pattering of surfaces,3 the physical trapping of cells in microscale wells,4 the use of dielectrophoretic (DEP) capture,5,6 and the hydrodynamic trapping of cells in microchannels.7,8 Powerful as these methods are, however, it remains difficult to use them to capture multiple cell types with positional control due to the limited number of clearly distinguishable surface interactions that are available. Although some research groups have reported success in patterning multiple cells on a single chip,6,8,9 the design flexibility of the patterns and number of the cell types that can be used are still limited. The patterning of more than two different cell types with arbitrary spatial relationships has not yet been achieved, but is highly desirable because it would enable the formation of complex heterotypic structures characteristic of tissues and organs. Such capabilities would also be useful for the © 2012 American Chemical Society
positioning of multiple cell types into arrays that could be used for drug screening. Recently, a new adhesion method has been developed in which living cells are directly derivatized with single-stranded DNA (ssDNA) through protein and carbohydrate modification10−13 or lipid insertion.14 The modified cells are then captured through hybridization to complementary sequences extending from a substrate. This concept has also been used successfully for the immobilization of vesicles and to control interactions between “artificial cells”.15 The DNA-mediated adhesion method is applicable to both nonadherent and adherent cells,11−14 does not cause major cell perturbations of cell function,12 and can generate densely packed arrays in a matter of minutes.12 When used with nonadherent cells, the DNA strands remain intact for 24 h or more in ordinary cell culture media,11b,12 and can be stabilized for long-term experiments through the addition of nuclease inhibitors. Adherent cells secrete extracellular matrix proteins and spread normally.11b By using several different DNA sequences, this method also offers a convenient way to capture multiple cellReceived: December 9, 2011 Revised: April 16, 2012 Published: April 18, 2012 8120
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types in predefined locations,11b providing heterogeneous 2D and ultimately 3D cell micropatterns for the purpose of studying intercellular communication and emergent behavior. What was needed to do this, however, was an efficient lithographic process that could be used to pattern multiple DNA capture strands with micrometer-level resolution. Here, we describe an improved patterning technique that can be used create arrays of multiple cell types for the study of cell signaling (Figure 1). In this approach, micropatterns composed
and amino groups at the ends, respectively. All oligonucleotides were obtained from Integrated DNA technologies (Coralville, IA). The sequences are as follows: A: HS−C6-5′-GTA ACG ATC CAG CTG TCA CT-3′−OH A′: H2N−C6-5′-AGT GAC AGC TGG ATC GTT AC-3′−OH B: HS−C6-5′-TCA TAC GAC TCA CTC TAG GG-3′−OH B′: H2N−C6-5′-CCC TAG AGT GAG TCG TAT GA-3′−OH C: HO−5′-ACT GAC TGA CTG ACT GAC TG-3′-C6−SH C′: HO−5′-CAG TCA GTC AGT CAG TCA GT-3′-C6−NH2 D: HS−C6-5′-ACT GAT GGT AAT CTG CAC CT-3′−OH D′: H2N−C6-5′-AGG TGC AGA TTA CCA TCA GT-3′−OH General Cell Culture Conditions. Jurkat cells were cultured in RPMI-1640 medium (Gibco/Invitrogen) supplemented with 10% (v/ v) fetal bovine serum (FBS, HyClone) and 1% penicillin/streptomycin (P/S, Sigma). FL-5.12 cells were grown in RPMI-1640 medium with 10% FBS, 1% P/S, 50 μM 2-metraptoethanol (Sigma), and 3.5 ng/mL murine recombinant IL-3 (Sigma). CHO cells were kept in F-12 (HAM) medium (Gibco) with 10% FBS and 1% P/S. For the cell communication experiment, the murine IL-3 gene was introduced to CHO cells by retroviral infection, and the gene encoding GFP was inserted into the vector pCDNA 3.1 and introduced to CHO cells by transient transfection.16 All cell lines were maintained at 37 °C in a 5% CO2 environment. Aluminum/PMMA Micropatterns on Glass Slides. A modification of the aluminum “lift-off” technique reported by Jackson and Groves was used.17 The starting substrate was an aldehydefunctionalized glass slide (SCHOTT Nexterion, Lousiville, KY) with a 100 nm aluminum (Al) layer deposited using a vacuum evaporator (401 vacuum system, Vecco). PMMA (MW: ∼996,000, Aldrich), which was dissolved in acetone (3.2% w/w), was spun on the slide at 2500 rpm for 30 s, followed by S1818 photoresist (PR) (Shipley) spincoated at 500 rpm for 10 s, and 2500 rpm for 30 s. The slide was then baked at 120 °C for 2 min to evaporate the solvent. The PR was exposed to UV with a photomask, and then developed with a 1:1 mixture solution of MicroDev developer and deionized (DI) water. The pattern was then transferred to the PMMA layer using oxygen plasma etching (Plasma-Therm PK-12 RIE, 150 W, 100 sccm O2). The Al layer was also etched with aluminum etchant (Air Products). To protect the aldehyde-glass surface from the aluminum etchant, the etching was stopped before the Al layer was completely removed (typical etching time: ∼1 min). The remaining Al layer was then removed with 0.05 M sodium hydroxide (NaOH). Finally, the PR layer on the PMMA was rinsed away with 2-propanol (IPA). DNA−NH2 Immobilization on the Slides. The attachment of DNA−NH2 to the aldehyde groups on the glass slides was based on a previously described protocol.12,14 After fabricating the aluminum/ PMMA micro patterns described above, chemical vapor deposition of trimethoxysilylpropanal (PSX1050-KG, UCT Specialties LLC) was performed on the glass slide at 60 °C for 1 h to maximize the number of aldehyde groups on the exposed glass surface. Then, 80 μM DNA− NH2 (sequence A−D) in phosphate buffered saline (PBS(−), Gibco) was placed on the pattern and incubated at room temperature (RT) for 15 min, followed by heating at 100 °C for 1 h to promote the amine-aldehyde condensation. The slide was next rinsed with 0.4% sodium dodecyl sulfate (SDS) in DI water, and immersed in a 65 mM solution of sodium borohydride (NaBH4, Sigma) in 1:3 PBS/ethanol to achieve the reductive amination. Finally, the unbound DNA−NH2 was washed away using 0.4% SDS in DI water, and the PMMA layer was removed with acetone. When patterning more than one type of DNA sequence, the PMMA patterning and DNA immobilization steps were simply repeated in the same manner. The DNA-patterned slides were stored under anhydrous conditions at RT and used within 2 weeks of preparation. DNA modification of live cells. The previously reported protocol for cell modification was followed.12 Briefly, 60 μL of a 0.39 μM solution of DNA with a thiol at the 5′ end (sequences A and D) was reacted with 0.2 mg of succinimidyl[(N-maleimidopropionamido)hexaethylene glycol] (NHS−PEO6−Maleimide) (Pierce) in 40 μL of
Figure 1. Self-assembly of heterogeneous cells on microscale patterns via DNA hybridization. (a) Cell surfaces are modified with singlestranded DNA (ssDNA, 20 bp) with an N-hydroxysuccinimidyl (NHS) ester at the 5′ end. The NHS-DNA modifies primary amino groups on the cell surface. (b) Multiple types of cells, which are modified with different DNA sequences, self-assemble on the complementary ssDNA micropatterns on a substrate. The DNA micropatterns are made by photolithography, and dimensions of the patterns vary from single cell resolution (∼10 μm) to whole wafer scale (typically 4−8 in.). (c) This technique can be used to prepare complex heterotypic cellular arrays that can be used to perform functional experiments depending on cell−cell communication or juxtaposition.
of multiple DNA sequences are defined using photolithography, allowing the formation of complex 2D patterns of ssDNA at single cell resolution (∼10 μm) over a wafer-sized area (∼4−8 in. in diameter). This technique was used to create a radial pattern of two different types of DNA-modified cells, which was then covered with an agarose hydrogel. This experimental setup was then used to track the ability of a secreted paracrine signal to induce cell proliferation over specified distances, paving the way for many future experiments that explore the effects of diffusible signals on stem cell differentiation and immune system function.
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MATERIALS AND METHODS
Oligonucleotide Sequences. Twenty base-pair sequences were designed for the modification of cell surfaces (A−D) and glass substrates (A′−D′). The sequences A−D and A′−D′ have thiol groups 8121
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DMSO to obtain 1 mL of 23.4 μM NHS-DNA in PBS. The target cells, which were rinsed twice with PBS before use, were then incubated with the NHS-DNA solution for 30 min at RT. The concentration of the cells was 5 × 106 cells/mL. CellTracker Blue, Green, or Orange (Invitrogen) was added to the NHS−DNA solution if fluorescent labeling was needed. Following this, the cells were washed twice with PBS containing 1% FBS, and resuspended in a fresh portion of 1% FBS in PBS at the concentration of 1 × 107 cells/mL. The cells were used for cell capture experiments within 2 h. Cell Capture by DNA Hybridization. The aluminum layer on the DNA-patterned glass slide was removed with 0.05 M NaOH. After a DI water rinse, the slide was immersed in PBS containing 1% FBS for 1−2 h to block the glass surface with proteins in the serum, which significantly lowered unwanted background cell binding. The slides were then placed in PBS solution in a Petri dish. The DNA-modified cells were dispersed on the slides using a micro pipet and incubated for 5 min for capturing the cells on the slide via DNA hybridization. Unbound cells were gently washed away by swirling the Petri dish containing the pattern and PBS. In the case of patterning two or three different types of cells, this series of dispersing and washing steps was sequentially repeated for each cell type. The captured cells were observed with a fluorescent microscope (Nikon, ECLIPSE E800). Culture of the Patterned Cells in Agarose. For the cell communication experiments, the patterned FL-5.12 and CHO IL-3 cells were immersed in an agarose hydrogel containing culture medium. Preparation of the agarose hydrogel was as follows: Ultralow gelling point agarose (Type IX-A, A2576, Sigma) was dissolved in PBS (2% (w/w)) and sterilized. The agarose solution was mixed with RPMI 1640 medium containing 12% FBS and 1% P/S at 1:1 ratio, and warmed to 37 °C. The glass slide with the patterned cells was then carefully immersed in 6 mL of the agarose/medium mixture in a 60 × 15 mm Petri dish and cooled at 4 °C for 30 min to form the gel. The cells were then incubated at 37 °C in a 5% CO2 environment for 72 h. For the control experiments in Supporting Informaion, Figure S4, CHO (+GFP) cells were used instead of CHO IL-3 cells, or 0.5 μg/ mL of mouse anti IL-3 antibody was added in the agarose. Observation of FL-5.12 Proliferation. Optical images of the patterned FL-5.12 and CHO IL-3 cells were recorded at 24 h time points using an inverted fluorescent microscope (IX71, Olympus). The number of FL-5.12 cells on the micro patterns was counted. For the cell viability assays, 1.5 μL of 4 mM calcein AM and 10 μL of 2 mM ethidium homodimer-1 (LIVE/DEAD viability/cytotoxicity kit for mammalian cells, Invitrogen) in 500 μL of PBS were applied to the 6 mL agarose gel containing the patterned cells, followed by incubation at 37 °C for 1 h.
Figure 2. Fabrication steps to form ssDNA micropatterns on a substrate. (a) A 100 nm aluminum layer is deposited on an aldehydecoated glass substrate by vacuum evaporation. Poly(methyl methacrylate) (PMMA) and S-1818 photoresist (PR) are then spin-coated onto the substrate, and the PR layer is photolithographically patterned with a first photomask. (b) The PR pattern is transferred to the PMMA and aluminum layers with oxygen plasma etching and 0.05 M NaOH wet etching, respectively. (c) After removing the PR with 2propanol (IPA), the first amino-ssDNA (DNA−NH2) is immobilized on the aldehyde-glass surface using reductive amination. (d) Unwanted DNA−NH2 on the PMMA surface is washed away together with the PMMA layer by acetone rinse. (e−h) A second DNA pattern is made using the same process as that for the first DNA pattern, except with a second photomask at step e and a second DNA−NH2 sequence at step g. Steps e−h can be repeated for patterning more than two different DNA sequences. (i) The remaining aluminum layer is finally removed with 0.05 M NaOH.
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RESULTS AND DISCUSSION The key challenge for multiplex patterning of different cells into user-defined arrays was the fabrication of multistrand DNA patterns with micrometer-sized features. Our approach used a combination of photolithography, aluminum (Al) liftoff,17 and DNA protection with poly(methyl methacrylate) (PMMA), as outlined in Figure 2. First, the Al, PMMA and photoresist (PR) layers were deposited on aldehyde-coated glass and patterned using standard microfabrication techniques (Figure 2a,b). Amine-ssDNA (DNA−NH2, 20 bases) was then immobilized on the exposed aldehyde glass surface with reductive amination (Figure 2c). An acetone rinse removed the PMMA layer together with unbound DNA−NH2 to leave the first DNA pattern (Figure 2d). A second sequence was then patterned in the same manner but with a different mask (Figure 2e−h). A key step for making the second DNA pattern was to spin a new PMMA layer directly over the DNA−modified surface (Figure 2e) before adding the photoresist. When this step was omitted, some DNA degradation was observed upon prolonged contact with the photoresist (Supporting Information, Figure S2). However, the protocols to coat the substrate with PMMA and
subsequently remove it with an acetone wash were not found to reduce the ability of the DNA to recognize the sequence complements extending from labeled cells (Supporting Information, Figure S1). To pattern more than two different DNA sequences on the substrate, steps e−h (Figure 2) were performed repeatedly. This iterative feature makes it possible to pattern large numbers of different DNA sequences, enabling the capture of many different cell types on the pattern. After iterative DNA patterning, the remaining Al layer was removed with NaOH (Figure 2i). Cell capture was first performed on simple patterns to examine the efficiency and specificity of the approach (Figure 3). In a first experiment, a single population of blue-labeled Jurkat T lymphoma cells were modified with sequence A, and then assembled onto micropatterns of complementary sequence A′ (Figure 3a−c). Little-to-no nonspecific cell capture was observed. The width of patterned features varied from 10 to 60 μm, and these features were represented with similar resolution in the patterns of captured cells. In a second version 8122
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Figure 3. Capture of DNA-modified cells on DNA micropatterns. All scale bars indicate 100 μm. (a−c) Jurkat cells modified with strand A were captured on A′ micropatterns. Line-to-space dimensions of the patterns were 10 μm/30 μm, 20 μm/40 and 40 μm/60 μm for a, b and c, respectively. (d−f) Two types of Jurkat cells, which were modified with either strand A (blue) or strand B (red), were captured on single cell sized array patterns (d), or in stripes and checker board patterns (e, f). (g) Three different types of cells, FL-5.12 (modified with C1, blue), Jurkat (C2, red) and CHO (C3, green), were assembled on stripe micropatterns. (h) Relative binding densities of the three cells. The patterned cells shown in part g were counted for evaluating the relative binding densities. The error bars represent standard deviation for three sets of the stripes.
(CHO IL-3 cells) that secrete IL-3 and express green fluorescent proteins (GFP). When cocultured, FL-5.12 cells would be expected to proliferate when they receive IL-3 from the nearby CHO IL-3 cells.18 In contrast, when the FL-5.12 cells are cocultured with CHO cells that express GFP but do not produce IL-3, the FL-5.12 should undergo apoptosis. This simple system thus provides a test scenario for the construction of devices that can study the communication between cells through diffusible signals. We used our lithographic fabrication technique to pattern 15 concentric ring patterns of FL-5.12 cells (R1-R15, width: 50 μm, d: 350−8800 μm) that surround a central group of patterned CHO cells (r: 250 μm) (Figure 4b). The patterned FL-5.12 and CHO IL-3 cells were embedded in 1% agarose hydrogels to prevent convective flow and cell movement in culture. This setup allowed the IL-3 molecules produced by the CHO IL-3 at the center to diffuse gradually outward toward the surrounding FL-5.12 cells, enabling their proliferation. Distance-dependent cell proliferation was observed after 72 h of culture (Figure 4c,d and Supporting Information Figure S3). We counted the number of the FL-5.12 cells every 24 h, and then calculated the growth ratio, Rgrowth, which was defined as the number of cells at a given time, t, divided by the number of cells before culture for each ring pattern (Figure 4f). The FL5.12 cells on R1-R8 proliferated and reached confluence (Rgrowth: >9 at 72 h). The growth ratio of the FL-5.12 cells on R9-R13, however, decreased as the distance increased from the
of this experiment, Jurkat cells were modified with A (blue) or B (red) sequences, and then captured on square array patterns (sequence A′ and B′) that were sized such that only one cell type was captured per pad (Figure 3d). Stripes and checker board patterns also captured cells with similarly high efficiency and selectivity (Figure 3e,f). In a third experiment, three different cell lines, FL-5.12 (a hematopoietic progenitor; sequence A, blue), Jurkat (B, red), and Chinese hamster ovary (CHO, C, green), were captured sequentially on a substrate patterned with lines of three different DNA sequences (A′, B′, and C′) (Figure 3g,h). Importantly, both nonadherent cells (Jurkat, FL-5.12) and adherent cells (CHO) could be specifically directed to the substrate. The relative binding density of these captured cells showed that ∼75% to ∼99% of cells were selectively retained by their complementary strands after washing. The lower binding density on the first DNA patterns was due to the greater number of the washing steps to which they were exposed. To examine the feasibility of this combination of techniques for performing functional assays on constructed cellular networks, we designed an experiment incorporating two cell types capable of communicating by a diffusible paracrine signal (Figure 4a). FL-5.12 cells are interleukin-3 (IL-3)-dependent cells, and they can live and proliferate only in the presence of IL-3 molecules. They undergo apoptosis in the absence of IL-3 in ∼36 h.18 We also prepared stably transfected CHO cells 8123
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Figure 4. Concept and design of a distance-based cell communication experiment using DNA-captured cells. (a) An interleukin-3 (IL-3) dependent cell line, FL-5.12, proliferates by receiving IL-3 molecules from gene-transfected CHO cells (+IL-3, +GFP) that are cocultured with the FL-5.12. However, FL-5.12 cells undergo apoptosis after 36−72 h when they are cocultured with CHO (+GFP) cells that do not produce IL-3. (b) For surface experiments, CHO IL-3 cells were patterned at the center of a ring pattern in which FL-5.12 cells were presented in surrounding rings. Fifteen FL-5.12 ring patterns (R1-R15), which varied from 350 to 8800 μm in radius (d1-d15), were used (bottom). The patterned CHO IL-3 and FL-5.12 cells were embedded in a 1% agarose gel layer containing culture medium (right). IL-3 molecules generated by CHO IL-3 diffuse to the patterned FL-5.12 cells. c) Image of the patterned CHO IL-3 and FL 5.12 cells before cell culture (t = 0 h). Green fluorescence indicates CHO IL-3 cells. (d) Image of the CHO IL-3 and FL-5.12 cell patterns after 72 h of culturing at 37 °C in 5% CO2. FL-5.12 cells on R1-R7, which were close to the CHO IL-3 core, proliferated and reached confluence. The proliferation rate decreased as the distance from the CHO IL-3 cells became larger (R9-R12), and almost no proliferation occurred in the outer rings (R13-R15). (e) Magnified images of FL-5.12 cells shown in B after applying calcein AM and ethidium homodimer-1. Green fluorescence indicated live cells and red fluorescence indicated dead cells. (f) The growth of the FL5.12 cells depended on their distance from the CHO IL-3 cells that were patterned at the center. The growth ratio was defined as (number of FL5.12 cells after culture)/(number of FL-5.12 cells before culture). (inset) The ratio of live cells, which was defined as (number of live cells)/(total number of cells), also decreased at R9-R15. The error bars represent the standard deviation for four replicate data sets. (g) Comparison of the growth ratios of FL-5.12 cells that were cocultured with (i) CHO IL-3 cells, (ii) CHO cells that do not produce IL-3, and (iii) CHO IL-3 cells with anti IL-3 antibodies in the medium. The error bars represent the standard deviation for four replicate data sets.
increasing distances from the CHO IL-3 cells (Figure 4f, inset), consistent with their growth ratio. As a control, we cocultured FL-5.12 cells with CHO cells that did not produce IL-3. In another control, FL-5.12 and CHO IL-
CHO IL-3 cells, and no proliferation occurred (Rgrowth:
