Design and sterilization of 3D-printed glass-bottomed multiwells for cell culture and immunostaining

Abstract 3D printers are increasingly found in wet laboratories, allowing researchers to print devices for their research needs. To make those devices suitable for cell culture, they should ideally be sterilized following protocols used in cell biology labs. Simultaneously, any sterilization protocol should maintain the mechanical stability of the printed device. Here we have studied two 3D printing materials, Poly-Lactic Acid (PLA) polymers and Acrylonitrile Butadiene Styrene (ABS)-like Photopolymer resins, which are prevalently used in 3D printing. We have subjected 3D-printed specimens of each material to UV light exposure, ethanol immersion and autoclaving, and have verified their mechanical stability at the macro- and nanoscales using tensile testing and nanoindentation. ABS-like resin (0.31 ± 0.07 GPa) remained mechanically viable after UV illumination (1.19 ± 0.02 GPa) or autoclaving (0.36 ± 0.05 GPa), but deteriorated when submerged in ethanol (0.01 ± 0.00 GPa). Nevertheless, sterilized ABS-printed devices probed to be toxic to cells, an effect that could not be prevented when switching to the inert Formlabs Grey resin. On the other hand, PLA specimens (1.22 ± 0.09 GPa) remained mechanically viable after ethanol (1.23 ± 0.15 GPa) and UV (1.21 ± 0.01 GPa) challenge but deteriorated in the autoclave (0.75 ± 0.20 GPa). This effect was prevented when switching to Heat-Treated PLA (HTPLA) polymers (1.07 ± 0.13 GPa). Sterilization of HTPLA-printed devices with a sequence of autoclave, ethanol and UV treatments deemed them suitable for long-term cell culture, as confirmed by measuring cell morphologies and doubling times (21.06 ± 4.66 h). Altogether, we provide here designs for in-house 3D-printing and assembly of glass-bottomed multiwells based on HTPLA and detail optimal sterilization protocols for their use in long-term cell culture and immunostaining. Key points • 3D printers can be used to create reusable culture devices • 3D-printed materials can be sterilized to be used as culture vessels • Standard sterilization already used in the lab can be used with 3D-printed materials