Fig 1.
Overview of the various software tools and their applications.
Geometrical objects can be encoded as python or C# files, as general use Standard Triangle Language (.stl) files, or as a sequence of machine specific commands (g-code). Internally, extrusion and movement commands are represented as a binarized gCodeCollection objects (.ptf) that can be edited and distributed into a number of parallel Petri dishes.
Fig 2.
Adaptation of an ultimaker original printer for culture dish printing.
A: Stage accepting an array of 35 mm cell culture dishes. B: Modified printhead. Yellow area: modified shroud to increase clearance to the stage and improve airflow. Blue area: a heater block that fits into the tissue culture dish. Red area: holder for a removable microswitch sensor, used to map the height of each dish. C: Printhead with microswitch sensor installed (orange). Another microswitch sensor, mounted on the stage (dark red) is used to calculate the height difference between the removable sensor and the nozzle.
Fig 3.
Optimization of conical wells for phase contrast optics.
A: A series of wells were printed with various wall slopes or cone aperture angles (left column). 3T3 cells within the wells were imaged using an automated phase contrast microscope. The tiled mosaic fields are shown in the middle column (scale bar: 500 μm). Red rectangles mark individual micrographs, that are shown in the right column (scale bar: 100 μm). The best optical image corresponds to a DMEM-PLA contact angle of 40°. B: Quantitative analysis of image contrast. For each well we assigned a contrast quality value, and data is pooled from n = 3 parallel sets of wells. Asterisks denote significant (p < 0.05) difference from the image obtained in cylindrical wells, error bars indicate standard deviation.
Fig 4.
Various 3D printed culture dish configurations.
A: Seven-well dish with a sloped wall to provide optimal optical quality. B: A double ring dish to control cell spreading and medium volume. C: A grid with 38 rectangular cells to store manually sorted cells. D: Phase contrast micrograph of a single PLA layer adhering to a tissue culture substrate, part of the cell sorter grid shown in panel C. Air bubbles indicate small areas with imperfect contact between the PLA layer and the underlying substrate. Scale bar: 500 μm.
Fig 5.
Cytotoxity assay for PLA exposure.
Cells were grown in the presence of PLA surfaces for two days after which their protein content was determined by an SRB assay. Data are shown as average of n = 4 parallel experiments for two cell ines (A: 3T3 cells, B: p31 cells) and the effect of treatment is expressed relative to untreated controls. Error bars represent standard deviation.
Fig 6.
Long-term biocompatibility assay with primary hippocampal neuron culture.
A: A triple ring dish used for cell culture. B: To achieve high cell density, cell spreading is restricted within the rings (diameter: 6 mm, height: 1.8 mm). A tiled image of phase contrast micrographs demonstrate the optical quality of the setup. Scalebar: 1000 μm. The red rectangle marks a region shown in higher magnification in panel C. D: Composite fluorescent image to evaluate cell death within the same region shown in panel C. Live cells are labelled by calcein (green), while dead cells are labelled by ethidium (red) and marked by arrows.