Fig 1.
Mod3D live-cell microscopy system.
(a) Examples of 22x50 mm 3-chamber holders in the universal 96-well plate format, with MSLA printed chambers, FDM printed holder, inserted magnets and reusable PLA/polystyrene lid. (b) Examples of the 6-chamber holders with different chamber designs. (c-e), Modularity and assembly of the system, where the holders and the lids are reusable.
Fig 2.
Examples of varieties of chamber designs in Mod3D.
(a,b) Examples of embossed alphanumeric labels on 4-well (750 μl per well) or 16-well (145 μl per well) chambers. (c) Round single well design (2.8 ml). (d) Example of glass bonding using RTV silicone at the bottom of the chambers. (e) Example of 8-well chambers (260 μl per well). (f) Bridged 2-well chambers in 22x50 mm format, shown bonded to 22x22 mm glass coverslips in (g). (h) Bridged 2-well chambers bonded to a 22x50 mm glass coverslip, with reusable plastic lid.
Fig 3.
Thermochromic chamber holder for live cell incubator stage.
(a) Left to right: the lid, chamber, and holder. (b) Assembly of the chamber and holder in the stage at 25°C with grey color. (c) Placement of the chamber and holder within the incubated temperature and gas control chamber at 37°C with color change to orange. (d) As in panel c, but temperature increased to 42°C (yellow).
Fig 4.
Sample live cell confocal microscopy data.
RPE1 cell expressing PARP1 chromobody as red fluorescent protein fusion. (a) After 24 hrs growth, immediately after 405nm laser irradiation in the region defined by yellow box. (b) 12 seconds later. Panel c, 25 seconds later, showing PARP1 chromobody recruitment to the site of induced DNA damage. Scale bar is 10um. Timestamp is seconds and milliseconds. 60X oil objective.
Fig 5.
Bespoke Mod3D perfusion chamber.
(a) Computer Aided Design (CAD) drawing of microfluidic paths and the 1:4 manifolds. (b) Chamber, culture lid and perfusion lid clamp. (c) A 22x22 mm coverslip is placed on top after cell culture sealed with silicone grease and clamped into place with the PETG clamp. Inlet/outlet aluminum tubing is bonded in place with RTV silicone.
Fig 6.
(a) 3D printed curing/sterilization chamber lined with aluminum foil tape with a 30W 385 nm LED flood lamp placed on top. (b) Alternate method of chamber sterilization in a UV curing chamber with rotating platform (Electrolite ELC-500). In both methods the chambers are placed in clear polypropylene bags.
Fig 7.
Growth and no toxicity or stress in Mod3D chambers.
(A) representative bright field images of cell growth of human TruHD cells. TruHD are growth contact inhibited, non-transformed lines. Scale is 1000um. (B) Fluorescent LIVE/DEAD assay comparing commercial chambers to Mod3D. Panel C, assay of mitochondrial ellipticity using NucBlue and Mitotracker dyes comparing commercial chambers to Mod3D. No significant differences were noted, p<0.5 by simple random sample and a Mann-Whitney nonparametric t-Test.
Table 1.
Cost comparisons of commercially available imaging chambers and the 3D printed microscopy chambers.