Letter pubs.acs.org/ac
Image-Based Single-Cell Sorting via Dual-Photopolymerized Microwell Arrays Tao Sun, Joseph Kovac, and Joel Voldman* Research Laboratory of Electronics, Department of Electrical Engineering and Computer Science Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States S Supporting Information *
ABSTRACT: We present a simple and cost-effective single-cell sorting method using two sequential photopolymerization steps that enables sorting based upon imaged phenotypes. The first photopolymerization step uses a thiolene-based resin with minimal autofluorescence to create an array of microwells to capture cells. The second photopolymerization uses (poly)ethylene glycol diacrylate to encapsulate undesired cells in a hydrogel, allowing for retrieval of the desired cell population using simple washing. We quantitatively characterize the method using fluorescently labeled cells and then applied the method to isolate cells based upon imaged fluorescence localization. The method is readily transferrable to other laboratories and will provide a facile route to sorting of cells based on imaged phenotypes.
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individual laboratories to perform analyses that are simply not possible today. Microtechnologies are well-poised to meet the challenge of low-cost cell manipulation. Numerous cell-sorting technologies have been developed, based on a variety of physical mechanisms, including electrokinetics,7,8 acoustophoresis,9 optofluidics,10−12 magnetism,13,14 hydrodynamic force,15,16 fluidic control,17 and adhesive-based schemes.18 A few microscale cell sorting devices have integrated spatiotemporal imaging,12,14 but they either cannot sort adherent cells12 or require manual one-by-one sorting.14 In this letter, we present an image-based single-cell sorting method that enables parallel cell sorting using a dualphotopolymerization scheme. Our approach enables simultaneously sorting multiple cells of interest following highresolution imaging with high purity using a method that requires only commonly available equipment at modest cost and should be able to be widely adopted by the community. Our overall approach is to spatially segregate cells using a microwell array created via photopolymerization, image them, identify the desired cells, and then remove the desired cells from the array by encapsulating all the undesired cells in a second photopolymer (Figure 1a). Because both the microwell
nderstanding how cells function is important for applications ranging from basic biology (e.g., understanding cell fate choices) to biotechnology (e.g., cell-produced biologics) to medicine (e.g., stem cell-based therapies). Our ability to understand cell function depends on the development of tools to manipulate and characterize cells. One important operation is that of sorting cells of interest from others. Fluorescence-activated cell sorting (FACS)1 and magnetically activated cell sorting (MACS)2 are perhaps the most common methods for sorting cells. FACS in particular has a beneficial combination of high throughput (up to ∼10 000 cells/s) while allowing multiparameter sorting. However, one significant limitation of FACS is in the range of phenotypes that it can screen, since FACS instruments do not image and only observe cells once. The inability to image makes FACS unable to sort based on morphological or localization-based phenotypes. This ability to obtain imaged information is important enough that commercial imaging flow cytometers3 and microfluidic systems4 have been developed to image cells in flow. However, the systems to-date cannot sort, and they are fundamentally limited to single observations of cells and thus cannot interrogate timelapse or long-term dynamics. Alternatively, commercial image-based cell sorting systems, e.g., Zeiss PALM MicroBeam5 and Cyntellect LEAP,6 add sorting to a microscopy platform. Unfortunately they are extremely expensive and not widely used. The ability to sort cells based upon imaged information at low cost would allow © 2013 American Chemical Society
Received: November 20, 2013 Accepted: December 19, 2013 Published: December 19, 2013 977
dx.doi.org/10.1021/ac403777g | Anal. Chem. 2014, 86, 977−981
Analytical Chemistry
Letter
is moved), which motivated use of a microwell array. Microwell arrays are a simple approach for large-scale single-cell trapping.12,20−23 Use of a microwell array has the further benefit that cells will be spaced by a known distance (the array spacing), which eases the subsequent sorting step. To further allow for high-resolution imaging, we adopted soft imprint lithography24 to fabricate the cell trapping microwell array on the surface of 40-mm-diameter coverslip-bottomed culture dishes (Electron Microscopy Sciences) from the thiolene-based resin NOA 81 (Norland optical adhesive). NOA 81 is conveniently applied to coverslips thus enabling high-numerical aperture microscopy and has minimal autofluorescence. Additionally, we found that it exhibited strong attachment to the overlying second photopolymer. We fabricated the microwell array by manually embossing a structured plastic master25 consisting of microposts (50 μm in diameter and depth, separated by 200 μm), and exposing to collimated ultraviolet (UV) light (Figure 1b-I and Figure S1 in the Supporting Information). Typical arrays consisted of 15 000 wells of ∼45 μm depth. The microwell array is compatible with oil-immersion, high numerical aperture (N.A, 1.4), and short working distance (W.D., 0.13 mm) objectives (Nikon, CFI Plan Apo VC60X) for imaging the cells with high resolution (Figure S2, Supporting Information). Three global alignment marks were created using a black-color marker on the bottom of the dish for subsequent alignment steps. We demonstrated the image-based sorting method using HeLa s3 cells. One line was stably infected with a GFP-CenpA reporter construct, while the other was infected with an mCherry-NFATc3 reporter construct.19 Cells were loaded into the dish and gradually trapped into microwells due to the gravitational settling (Figure 1b-II). The untrapped cells were rinsed away by washing. The next step in the process was to image the cells in the array. We scanned the microwell array under microscopy to image the cell array under phase and fluorescence. Desired cells (e.g., green cell in Figure 1b-III) can be determined manually or by automated imaging algorithms. After recording the positions of all the cells of interest, we initiated the sorting step. Here we wanted a sorting method that was parallel rather than serial, in order to be able to remove many desired cells at once. Since the microwell array was situated in a culture dish, a method that would be compatible with standard pipetting would be advantageous in avoiding enclosed microfluidics. We thus
array and the encapsulant are formed from photopolymers, the method is termed dual-photopolymerization cell sorting.
Figure 1. (a) Method overview. (b) Detailed steps of image-based single-cell sorting method via dual-photopolymerized microwell arrays. Trapping wells are created by the first photoploymerization process (step I). They are used to trap individual cells (step II). Sorting wells are created by the second photopolymerization process (step V). They are larger than the trapping wells and layered on top of the trapping wells. They are used to isolate the desired cells to be sorted out (step VI).
We previously developed a method that used a single photopolymerization step to sort adherent cells from culture dishes.19 That method, however, was not suitable for use with nonadherent cells nor with high-resolution imaging. Imaging nonadherent cells without losing registration requires compartmentalizing them (since they move around when the container
Figure 2. Whole-cell fluorescence-based cell sorting of mixtures of mCherry+ and GFP+ HeLa cells. (a) Red/green fluorescence image of trapped cells in the photopolymerized microwell array created by the first photopolymerization. (b−d) Phase (top) and fluorescence (bottom) close-up images of (b) a desired GFP+ cell in the midst of undesired mCherry+ cells, (c) after the second photopolymerization, showing that the desired GFP + cell is isolated from undesired mCherry+ cells by the PEGDA polymer sorting well, and (d) after the desired cell is removed via washing. (e) View of 1 mm × 1.4 mm region of the array showing trapping wells and sorting wells. 978
dx.doi.org/10.1021/ac403777g | Anal. Chem. 2014, 86, 977−981
Analytical Chemistry
Letter
trapping wells (photopolymerized optical adhesive) and the sorting wells (photopolymerized PEGDA). To assess the recovery and purity of sorted cells, we sorted GFP-expressing cells from a 1:100 GFP/mCherry mixed culture. We targeted microwells that contained desired GFPexpressing cells, and after sorting, we replated the sorted cells into one well of a 96-well plate and monitored their growth in culture. From the 41 GFP-expressing cells originally selected, we observed 32 colonies of GFP-expressing cells and 3 colonies of mCherry-expressing cells on d 3 of culture (Figure S4, Supporting Information), corresponding to a recovery of 78% and a purity of 91%. The cell loss (∼22%) likely occurred from a combination of dish washing, supernatant filtering, cell centrifuging, and replating steps. We have repeated the sortings multiple times (n > 3) and obtained consistent sorting efficiency and cell recovering rate. Overall, the enrichment of desired cells is almost 1000-fold, which allows for immediate application in enriching minority cells. To demonstrate a phenotypic sort that cannot be achieved by FACS, we sorted cells based on fluorescence localization. We stained the GFP-expressing cells with SYTO 82 nucleic acid dye and mixed them with the mCherry-expressing cells at a ratio of 1:100. The SYTO-stained GFP-expressing cells (desired) showed whole-cell red fluorescence because the orange-red SYTO dye stains both DNA and (cytoplasmic) RNA, while the mCherry-expressing cells (undesired) showed red fluorescence only within the cytoplasm because of the fusion to NFATc3, which is normally resident in the cytoplasm. We used microscopy to distinguish cells with whole-cell red fluorescence from cells with cytoplasmic red fluorescence, using green fluorescence as a “ground truth” for verification (Figure 3a). We then initiated the sorting. Figure 3b shows a closeup of the array demonstrating that the desired SYTO-stained GFPexpressing cell was removed, while the mCherry-expressing cells remained. This experiment successfully demonstrates that dual photopolymerization allows for sorting based on imaged information.
sought a method that would allow all desired cells to be removed by washing while encapsulating nondesired cells. We chose to use photopolymerizable hydrogels because they are cell-compatible26−28 and can be configured in real-time simply by changing the polymerization mask. We used (poly)ethylene glycol diacrylate (PEGDA) for our second photopolymerization. We introduced a prepolymer solution to the array dish (Figure 1b-IV), and using a process adapted from prior work,19 we generated a photomask image file consisting of black dots (180 μm diameter) at desired locations via a Matlab script (Mathworks), with each black dot corresponding to the position of a desired cell. Three alignment circles corresponded to the positions of three global alignment marks on the bottom of the dish (Figure S3, Supporting Information). We printed the image onto a standard inkjet transparency film (Office Depot) using an inkjet printer (HP Deskjet 6540) and then aligned the mask under the array dish by hand under a microscope (Stemi 2000-C, Zeiss) (Figure 1bIV). We were able to align the masks within 1 min with typical alignment accuracies of ∼10 μm. After the alignment, the device was exposed to UV light for the second photopolymerization (Figure 1b-V). This cross-linked the PEGDA prepolymer into a hydrogel in all unmasked regions, encapsulating the undesired cells. The black dots on the mask shielded the desired cells from the UV exposure, leaving unpolymerized prepolymer around those cells and thus making them available for removal by washing. Finally, we rinsed the dish with cell culture medium to retrieve the desired cells (Figure 1b-VI). To illustrate and quantitatively characterize the sorting method, we used simple whole-cell fluorescence-based sorting. Although such sorts can be performed using FACS, they are useful to quantitatively characterize the method. Figure 2a shows a typical cell trapping result where we loaded a 1:1 ratio of GFP/mCherry-expressing cells to achieve approximately 90% well-filling efficiency. After washing, very few (
