Staurosporine (Sigma-Aldrich S5921) was prepared by serial dilution from a 10 mM DMSO stock. Cell lines without endogenous fluorescent markers were labeled with CellTracker Deep Red or Blue CMHC dyes according to manufacturer protocols (ThermoFisher Scientific C34565 and C2111 respectively). Cell viability was measured via microplate-based PrestoBlue assay according to manufacturer instructions (ThermoFisher Scientific A13261).

Reconstituted Tissue Fabrication

Todhunter et al., 2015 Todhunter M.E.

Jee N.Y.

Hughes A.J.

Coyle M.C.

Cerchiari A.

Farlow J.

Garbe J.C.

LaBarge M.A.

Desai T.A.

Gartner Z.J. Programmed synthesis of three-dimensional tissues. Weber et al., 2014 Weber R.J.

Liang S.I.

Selden N.S.

Desai T.A.

Gartner Z.J. Efficient targeting of fatty-acid modified oligonucleotides to live cell membranes through stepwise assembly. 20 -3’, see Microfluidic flow cells were constructed by sandwiching aldehyde-silanized glass slides (Schott 1064874) against a polydimethylsiloxane (PDMS) membrane gasket (0.01” thick, SSP M823) cut with a craft cutter (Silhouette). Prior to sandwiching, through-holes in the top slide were made using 20 passes of a 50 W etching laser at 100% power, 15% speed, 350 pulses per inch (VLS3.5, Universal Laser Systems). Fiducial marks were etched into both glass slides to aid alignment by light microscopy before cell patterning. Etched slides were used as substrates for DNA-programmed assembly of cells (DPAC), as detailed previously (). To summarize, after etching, amine-modified oligonucleotides (5’-amine-X-3’, see Key Resources Table ) were printed onto the slides using a microfluidic cantilever (NanoEnabler, Bioforce Nanosciences) and covalently attached by reductive amination. Printing locations were specified in bitmap files. Slides were treated with hydrophobic silane and blocked for 1 hr in 3% bovine serum albumin in PBS before being assembled against the PDMS gasket. Immediately prior to sandwiching, PBS was added to gaps in the gasket to prime flow cells and reduce the tendency to trap bubbles between the slides. Slight pressure was applied to the sandwich using a microarray hybridization cassette (AHC1X16, ArrayIt). The positions of through-holes in the top glass slide and voids in the PDMS gasket were matched to the dimensions of the cassette such that 16 flow cells could be independently addressed through pairs of through-holes.

20 -T 60 -X 20 -3’ followed by a 3’-lipid-Y 20 -5’ “co-anchor”, see Todhunter et al., 2015 Todhunter M.E.

Jee N.Y.

Hughes A.J.

Coyle M.C.

Cerchiari A.

Farlow J.

Garbe J.C.

LaBarge M.A.

Desai T.A.

Gartner Z.J. Programmed synthesis of three-dimensional tissues. Weber et al., 2014 Weber R.J.

Liang S.I.

Selden N.S.

Desai T.A.

Gartner Z.J. Efficient targeting of fatty-acid modified oligonucleotides to live cell membranes through stepwise assembly. 6 cells/ml were introduced to flow cells by gentle pipetting on top of one of the pair of through-holes and adhered to DNA spots on the glass. A further round of cellular assembly was used to generate clusters of 5-8 cells at each DNA spot. After cell patterning, liquid gel precursor was introduced in two aliquots of 20 μl per flow cell and the cassette placed at 37° C for 20 min to set the precursor. Reconstituted tissue gels consist of a composite of fluorescently-labeled collagen I fibers in matrigel. To prepare the gel precursor, 200 μl of ∼8.5 mg/ml rat tail collagen I in 0.02 M acetic acid (Corning 354249) was labeled using 5 μl of 1 mg/ml Alexa Fluor 555- or 647-NHS ester in DMSO (ThermoFisher Scientific A20009 and A20006; chosen to avoid spectral overlap with cell labels in a given experiment) that was added immediately prior to neutralization with 10 μl 20x PBS and 4 μl 3 M NaOH on ice. After 10 min on ice, 70 μl of this collagen stock was added to a second stock consisting of 415 μl of ∼ 9 mg/ml matrigel (Corning 354234) and 15 μl of Turbo DNase (ThermoFisher Scientific AM2238). This 500 μl precursor solution was sufficient to build reconstituted tissues in 8-10 flow cells. Cells to be assembled on the top and bottom flow cell walls were lifted from plates using a PBS wash followed by 0.05% trypsin and labelled with lipid-modified oligonucleotides (5’-lipid-Y-T-X-3’ followed by a 3’-lipid-Y-5’ “co-anchor”, see Key Resources Table ) as previously described () (with the exception of Caco2 cells, see below). With the flow cell cassette on ice, cells in suspensions of ∼10 x 10cells/ml were introduced to flow cells by gentle pipetting on top of one of the pair of through-holes and adhered to DNA spots on the glass. A further round of cellular assembly was used to generate clusters of 5-8 cells at each DNA spot. After cell patterning, liquid gel precursor was introduced in two aliquots of 20 μl per flow cell and the cassette placed at 37° C for 20 min to set the precursor. Reconstituted tissue gels consist of a composite of fluorescently-labeled collagen I fibers in matrigel. To prepare the gel precursor, 200 μl of ∼8.5 mg/ml rat tail collagen I in 0.02 M acetic acid (Corning 354249) was labeled using 5 μl of 1 mg/ml Alexa Fluor 555- or 647-NHS ester in DMSO (ThermoFisher Scientific A20009 and A20006; chosen to avoid spectral overlap with cell labels in a given experiment) that was added immediately prior to neutralization with 10 μl 20x PBS and 4 μl 3 M NaOH on ice. After 10 min on ice, 70 μl of this collagen stock was added to a second stock consisting of 415 μl of ∼ 9 mg/ml matrigel (Corning 354234) and 15 μl of Turbo DNase (ThermoFisher Scientific AM2238). This 500 μl precursor solution was sufficient to build reconstituted tissues in 8-10 flow cells.

The flow cell cassette was then disassembled gently, and the slide sandwich submerged in the appropriate cell media at room temperature. A razor blade was used to gently pry apart the glass slides. Reconstituted tissues consisting of cell clusters carried along with the ECM gel typically floated spontaneously into the media or could be gently detached from one of the glass slides with a micro-spatula (Fine Science Tools 110089-11). Floating tissues were then manually cut out using either a biopsy punch or razor blade, or by laser microdissection (Zeiss PALM MicroBeam). Finished tissues were transferred to glass coverslip-bottomed 24-well plates (Greiner) using a P1000 pipet trimmed to a ∼7 mm diameter. If reconstituted tissues were intended to undergo folding, the glass in each well was coated with 1% agarose in PBS prior to adding tissues to prevent them from adhering. For imaging studies of collagen strain/alignment, cell migration, and non-folding controls, or prior to microdissection, reconstituted tissues were encouraged to adhere to the bottom of coverslip wells by 10 min 37° C incubation in a semi-dry state (with media temporarily withdrawn).

Rather than being assembled by DPAC, a semi-confluent layer of Caco2 cells was assembled at the lower tissue surface by pre-mixing them at 4 x 106 cells/ml in gel precursor such that they settled onto the bottom of the flow cell prior to setting at 37° C.

Reconstituted tissues that contained HUVECs were cultured in EGM-2 with 200 ng/ml each of IL-3, stromal-cell derived factor 1a (SDF-1a) and stem-cell factor (SCF) to encourage lumenization. Tissues with a single passenger cell type were cultured for 12 hours in the appropriate fibroblast medium, and transferred to the passenger cell type’s medium thereafter. 3-cell type reconstituted tissues (containing MEFs, HUVECs, and Caco2 cells) were similarly transferred to 50:50 HUVEC:Caco2 media after 12 hours. We anticipate that significant optimization of media conditions will be required depending on the targeted endpoints relevant to cell types of interest.