Microfluidic chambers using fluid walls for cell biology

Abstract

Many proofs of concept have demonstrated the potential of microfluidics in cell biology. However, the technology remains inaccessible to many biologists, as it often requires complex manufacturing facilities (such as soft lithography) and uses materials foreign to cell biology (such as polydimethylsiloxane). Here, we present a method for creating microfluidic environments by simply reshaping fluids on a substrate. For applications in cell biology, we use cell media on a virgin Petri dish overlaid with an immiscible fluorocarbon. A hydrophobic/fluorophilic stylus then reshapes the media into any pattern by creating liquid walls of fluorocarbon. Microfluidic arrangements suitable for cell culture are made in minutes using materials familiar to biologists. The versatility of the method is demonstrated by creating analogs of a common platform in cell biology, the microtiter plate. Using this vehicle, we demonstrate many manipulations required for cell culture and downstream analysis, including feeding, replating, cloning, cryopreservation, lysis plus RT-PCR, transfection plus genome editing, and fixation plus immunolabeling (when fluid walls are reconfigured during use). We also show that mammalian cells grow and respond to stimuli normally, and worm eggs develop into adults. This simple approach provides biologists with an entrée into microfluidics.

Keywords: fluid walls; interfacial tension; microfluidics; sessile drops; tissue culture.

Fig. 1.

Reverse printing. (A) Principle. A Petri dish is filled with DMEM plus 10% FBS, most medium is removed, and the residual thin film is overlaid with FC40 (shown in green). A hydrophobic stylus is lowered to touch the dish and to bring FC40 to the surface. As the stylus moves horizontally, medium is pushed aside and FC40 takes its place. This creates a track of FC40 pinned to the substrate and a liquid wall of FC40 dividing the aqueous layer. Drawing more lines creates a grid. (B) A 32 × 32 grid made in ∼4 min. After printing, 70 nL of yellow or blue dye is added to each chamber. (C) A high-density grid made with a thin stylus (73% surface covered by medium). (D) Adding and subtracting medium. (i) The contact angle is <θ_E (∼70°). (ii) Medium can be added without altering the footprint until θ_A is reached (θ_A > θ_E). (iii) Medium can be removed without altering the footprint until θ_R is reached (<3°). (iv) If θ_A is exceeded, the pinning line breaks and chambers merge. (v) Within limits imposed by θ_A and θ_E, grids are used like conventional plates; aqueous liquids are pipetted into (or out of) chambers through FC40 instead of air. Here, 1 to 3 µL of dye was added to chambers initially containing 0.1 µL of medium. Note that the maximum contact angle in the square drop with 3 µL was >70°. (E) Stylus width determines wall width. Lines were made using styli with wider and narrower tips than in B, and regions between chambers were imaged.

Fig. 2.

Some different ways of making grids. (A) FC40 isolates chambers effectively. A 1.5 µL portion of medium ± E. coli was pipetted manually into every second chamber in an 8 × 8 grid (pattern shown in cartoon), with 2 × 2 mm chambers. After incubation (24 h; 37 °C), a phase-contrast image was collected. Bacteria grew only in inoculated drops (seen as aggregates in chambers containing exhausted, slightly yellow media), and the rest remained sterile (slightly-pink chambers). (Inset) Aggregates and granularity indicate presence of bacteria. (B–D) Various overlays and substrates yield stable pinning lines. Films of media on polystyrene (uncoated except in D) or on glass (C) were overlaid with 2 mL of FC40 (or other oils in B), grids were created, and phase-contrast images were collected. (E) Preparing grids with different starting volumes (2.25 × 2.25 mm chambers). (i–iii) Three dishes were covered with medium plus blue dye, FC40 was added, four 20-mm lines were drawn to create one central square in each dish (volume ∼ 10 µL), and 0, 32, or 64 µL was pipetted into squares. (iv–vi) Subdivision yields grids.

Fig. 3.

Adding liquids discontinuously and continuously. (A) Discontinuous. (i–iii) The pipet is lowered (black arrow), and the pump ejects liquid (white arrow) that eventually merges with the chamber. (iv, v) After stopping the pump, the pipet is raised and moves to the next chamber. (B) Frames from Movie S3 illustrating that no red dye moved up through a liquid bridge to the pipet. Note that contact angles at the centers of chamber edges are ∼90°. (C) Lack of carryover of bacteria during discontinuous delivery (2 × 2 mm chamber, ∼150 nL). (i) A 300-nL portion of LB with or without ∼20,000 E. coli was added manually to every second chamber, and then 500 nL of LB was added to all chambers by discontinuous delivery. (ii) After 3 d at 20 °C, imaging shows that bacteria (white) grew only in inoculated chambers. (D) Continuous (scanning). (i–iii) The pipet maintains a constant height (450 µm) above the substrate as it traverses (black arrow), continuously ejecting liquid (white arrow). (iv) Liquid is delivered to the chamber. (v) Continuing traverse breaks the liquid bridge (maximum final chamber height 380 µm). (E) Frames from Movie S4. The tip scans (15 mm/s) along a serpentine path, delivering ∼70 nL of medium plus blue dye to each chamber. Insets show 4× magnifications. (F) No carryover of fluorescent beads between chambers during scanning (16 × 16 grid, 2 × 2 mm chambers, ∼150 nL). (i) A 300-nL aliquot of medium with (+) or without (−) 9,000 fluorescent beads (1-µm diameter) was added manually to every second chamber in the grid. A total of 500 nL of medium was added by scanning to each chamber. (ii) Phase-contrast (Left) and fluorescent (Right) images showing that no beads were carried over between chambers.

Fig. 4.

Biocompatibility (2 × 2 mm chambers, initial volume 150 nL). (A) Growth and feeding. (i) A total of 250 NM18 cells in 800 nL were added to chambers by scanning, grown (24 h), fed (800 nL removed and 800 nL fresh medium added), and imaged. (ii) High-power view of selected region. (Magnification: 20× objective.) (iii) Cells were regrown (24 h) and reimaged, revealing that the cells grow normally. (B) Trypsinization and replating. (i) A total of 500 HEK cells in 500 nL are plated by scanning, grown (24 h), and imaged. (ii) After trypsinization, 1 µL is retrieved, and the chamber is reimaged; most cells have been removed. (iii) Retrieved cells were deposited in a new chamber, and this chamber was imaged. (iv) After growth (24 h), imaging confirmed successful cell transfer. (C) Cryopreservation. HEK cells are cryopreserved in grids for 3 wk at −80 °C, thawed, and regrown (24 h). Pinning lines and cells survive freezing and thawing. (D) Creating fluid walls around growing cells. (i) HEK cells (2 × 10⁶) are plated, grown (24 h), and imaged. (ii) A grid was prepared around these adherent cells, and the same region of the dish was reimaged. A fluid wall divides the field; cells 1, 2, and 3 remain in their original positions as others in the path of the stylus are moved. (E) C. elegans develops and responds to osmotic stress normally. (i) Frame from Movie S5 illustrating worms living in a grid. (ii) Single eggs in 500 nL of S medium were deposited manually in chambers and fed daily; after 5 d, the resulting adult was imaged. (iii) Individual trauma-sensitive worms (strain CB7317) in 500 nL of S medium were deposited manually in chambers, and 500 nL of medium plus bacteria with or without 600 mM NaCl was added; after 2 d, worms were imaged. This strain expresses GFP in response to osmotic stress, and worms exposed to 300 mM NaCl fluoresce green. (Insets) Fluorescence images.

Fig. 5.

Rebuilding fluid walls and destroying cell walls (2 × 2 mm chambers). The 6-cm dishes were mounted in positioning rings (tightly fitting circular sleeves enabling a dish plus ring to be removed from a printer and remounted so that walls can be rebuilt in the same place). (A) Breaking and remaking fluid walls during immunolabeling. (i) NM18 cells were plated by scanning, grown, and fixed; fluid walls were broken, all cells batch permeabilized with Triton X-100, and walls remade. Fluid walls survive fixation but are destroyed by emptying the dish of FC40 and washing with PBS (some FC40 remains pinned to the dish). After returning the dish to the printer and overlaying FC40, fluid walls are rebuilt. (ii) Cells respond to TGF-β1 as expected. The workflow involves cycles of making and destroying fluid walls (SI Appendix, Fig. S5); when walls are present, individual chambers are treated differently, and when walls are absent, all cells in all of the different parts of the dish are treated similarly. Cells were seeded by scanning in chambers, grown with (+) or without (−) TGF-β1, and then fixed. Next, walls were destroyed, fixative was washed away, cells were permeabilized, walls were rebuilt, phalloidin or anti-vimentin (both conjugated with Alexa 488) was added to selected chambers, walls were destroyed, cells were washed, walls were rebuilt, DAPI was added, walls were destroyed, cells were washed, walls were rebuilt, and phase and fluorescent images were collected. TGF-β1 induces actin bundling and increases vimentin expression (detected by phalloidin labeling and immunolabeling, respectively). (B) Lysing cells and RT-PCR. (i) HEK-293 reporter cells were plated by scanning in chambers and then grown (24 h), medium with (+) or without (−) TNF-α (10 ng/mL) was added, cells were regrown (24 h) to allow the cytokine to induce GFP expression before washing and lysis, and lysates were transferred from chambers to microcentrifuge tubes for assessment of levels of GFP mRNA by RT-PCR. (ii) Pinning lines survive lysis. (iii) Phase-contrast (Top) and fluorescence images (Bottom) show TNF-α induces GFP expression. RT-PCR also showed that TNF-α increases GFP mRNA levels 205-fold (lower and upper bounds: 174- and 241-fold), normalized relative to the control (lower and upper bounds: 0.63- and 1.6-fold; n = 4).

Fig. 6.

A complex workflow: deriving a NM18 cell clone with a mutated Casp6 gene using CRISPR-Cas9. (A) Workflow. Cells were transfected with empty vector (encoding Cas9 plus puromycin resistance) or with plasmid 880 or 881, which additionally encode a guide RNA (gRNA) targeting different parts of Casp6. (B) Cloning. After transfection, puromycin selection (SI Appendix, Fig. S6), and expanding the cells conventionally, the printer delivered ∼0.2 cells (by scanning) to each chamber; images show the single cell divides over 8 d. Initially, 42 of 256 chambers (40 expected from Poisson distribution) contained one cell; after 8 d, 30 had colonies (71% cloning efficiency). (C) Genotyping. After picking colonies (as in Fig. 4B), the printer delivered the cells to a microcentrifuge tube. Cells were then expanded conventionally, DNA was purified, the Casp6 region was amplified by PCR and cloned in bacteria, and genotypes were determined by sequencing. Clones derived from transfections with plasmids 880 and 881 have deletions (del) in expected regions, unlike those receiving empty vector. Immunoblotting confirmed the phenotype (SI Appendix, Fig. S6).