On the intrinsic sterility of 3D printing - PubMed (original) (raw)

On the intrinsic sterility of 3D printing

Russell Y Neches et al. PeerJ. 2016.

Abstract

3D printers that build objects using extruded thermoplastic are quickly becoming commonplace tools in laboratories. We demonstrate that with appropriate handling, these devices are capable of producing sterile components from a non-sterile feedstock of thermoplastic without any treatment after fabrication. The fabrication process itself results in sterilization of the material. The resulting 3D printed components are suitable for a wide variety of applications, including experiments with bacteria and cell culture.

Keywords: 3D printing; Cell culture; Methods; Microbiology; PLA; Pasteurization; Polylactic acid; Sterile technique.

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Conflict of interest statement

Author Emily Tung works for Pivot Bio, a startup company affiliated with the QB3 incubator, which is itself affiliated with UCSF.

Figures

Figure 1

Figure 1. Temperature and duration of various sterilization processes.

Temperatures and durations for various methods of sterilization compared to fused deposition modeling (FDM) 3D printing. The extrusion process most closely resembles pasteurization, in which non-sterile liquid is forced through a narrow, heated tube. High-temperature, short-time (HTST) pasteurization is used for milk, fruit juices and other beverages and ingredients. Ultra-high temperature (UTH) processing is used to produce products such as shelf-stable milk that do not require refrigeration. Stove- top pasteurization (30 min at 63 °C) is indicated as “stovetop” pasteurization. Thermization, a process used to extend the shelf life of raw milk that cannot be immediately used, such as at cheese making facilities. Typical autoclave cycles using prevacuum, and gravity displacement are indicated as “prevacuum” and “gravity,” respectively. A typical “flash” sterilization cycle for a gravity displacement sterilizer is also indicated. Pasteurization processes are indicated in black, autoclave processes in red, and thermization in orange.

Figure 2

Figure 2. Preliminary experimental results.

Growth after 96 h at 37 °C in a shaking incubator. The beaker labeled (A) contains LB media inoculated with PLA plastic extruded from the printer nozzle at 220 °C. The beaker labeled (B) contains LB media inoculated with a segment of unextruded PLA plastic filament from the same spool. The beaker labeled (C) contains uninoculated LB.

Figure 3

Figure 3. Mouse macrophage growth in the presence of 3D printed parts.

Macrophages derived from mouse bone-marrow after incubation with 3D printed parts that had been treated with UV (A), without UV treatment (B), and treated with UV after handling and before incubation (C) and a control set of cells grown without 3D parts (D). Photos representative of three replicates in two independent experiments. Cell size, morphology and confluency were determined to be consistent across all experimental groups.

Figure 4

Figure 4. A 3D printed motility assay device.

A custom device for a motility assay fabricated using 3D printing. The device was found to be sterile without autoclaveing if contamination during post-fabrication handling is avoided.

Figure 5

Figure 5. Design and fabrication of test parts.

A very simple model of a cylinder was created in OpenSCAD and exported in STL format. (A) The G-code toolpath visualization of test part in Cura. The slicing engine was set to a 0.4 mm wall width (equal to the diameter of the nozzle), cooling fans inactive, no infill, a top and bottom layer height of zero, and a spiralized “Joris Mode” outer wall. (B) Test parts were then 3D printed on abraided and flamed aluminum foil at 220 °C with a feed rate of 50 mm/s.

Figure 6

Figure 6. Independent replication of results.

After 48 h, only the positive control (A) was contaminated. Printed cylinders in LB did not appear to contaminate the media (C and D). Tube (B) contains uninoculated media.

Figure 7

Figure 7. Test on solid media.

A total of 10 µl of each AYE tube (positive control, PLA plastic and negative control) was struck out on Charcoal Yeast Extract solid media and incubated at 37 °C to grow for 24 h. Growth revealed that the PLA test part (A) appeared to contain a different bacterial species than the positive control tube (B). Media from the negative control part was also plated (C). Using a light microscope, both bacterial growths appear coccoid, with the yellow colonies forming clumps more often. Experiment was repeated with parts from UC Davis and Michigan State, plus controls. Contamination was not observed.

Figure 8

Figure 8. Test in Terrific Broth.

3D printed parts from UC Davis with and without UV treatment (B and C) were suspended in sterile Terrific Broth supplemented with potassium salts, along with a negative control (A) and a positive control (D). After 24 h at 37 °C, no growth was observed for parts treated with UV. Tube (F) contains a part from Michigan State after 48 h of incubation in Terrific Broth, along with negative and positive controls (E and G). No growth was observed 96 hours after inoculation.

Figure 9

Figure 9. Test in anerobic conditions.

After two weeks in anaerobic chamber at 37 °C in “meat broth,” a non-UV treated part from UC Davis exhibited evidence of growth. All other parts were limpid, aside from the positive control. Contaminated media was plated on BHI+blood agar overnight (see Fig. 7), and 16S rRNA sequencing was performed on resulting colonies.

Figure 10

Figure 10. Test with alternative 3D printing technology (Objet Eden 260).

A group of cylinders were printed on an Objet Eden 260. 24 cylinders were transfered directly from the printing plate to culture tubes by scraping them from the build plate with the open tube. Two cylinders were removed with an ungloved hand to act as positive controls. Tubes were incubated for 96 h at 37 °C in LB media, revealing one contaminated tube.

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