A 3D-printed microbial cell culture platform with in situ PEGDA hydrogel barriers for differential substrate delivery

Additive manufacturing, or 3D-printing techniques have recently begun to enable simpler, faster, and cheaper production of millifluidic devices at resolutions approaching 100-200 μm. At this resolution, cell culture devices can be constructed that more accurately replicate natural environments compared with conventional culturing techniques. A number of microfluidics researchers have begun incorporating additive manufacturing into their work, using 3D-printed devices in a wide array of chemical, fluidic, and even some biological applications. Here, we describe a 3D-printed cell culture platform and demonstrate its use in culturing Pseudomonas putida KT2440 bacteria for 44 h under a differential substrate gradient. Polyethylene glycol diacrylate (PEGDA) hydrogel barriers are patterned in situ within a 3D-printed channel. Transport of the toluidine blue tracer dye through the hydrogel barriers is characterized. Nutrients and oxygen were delivered to cells in the culture region by diffusion through the PEGDA hydrogel barriers from adjacent media or saline perfusion channels. Expression of green fluorescent protein by P. putida KT2440 enabled real time visualization of cell density within the 3D-printed channel, and demonstrated cells were actively expressing protein over the course of the experiment. Cells were observed clustering near hydrogel barrier boundaries where fresh substrate and oxygen were being delivered via diffusive transport, but cells were unable to penetrate the barrier. The device described here provides a versatile and easy to implement platform for cell culture in readily controlled gradient microenvironments. By adjusting device geometry and hydrogel properties, this platform could be further customized for a wide variety of biological applications.