Acute brainstem slice preparations in vitro have advanced understanding of the cellular and synaptic mechanisms of respiratory rhythm generation, but their inherent limitations preclude long-term manipulation and recording experiments. Here, we developed an organotypic slice culture preparation containing the preBötzinger complex (preBötC), the core inspiratory rhythm generator of the ventrolateral brainstem. We measured bilateral synchronous network oscillations, using calcium-sensitive fluorescent dyes, in both ventrolateral (presumably the preBötC) and dorsomedial regions of 7-43 days in vitro (DIV) slice cultures. These calcium oscillations appear to be driven by periodic bursts of inspiratory neuronal activity, because whole-cell recordings from ventrolateral neurons in culture revealed inspiratory-like drive potentials and no oscillatory activity was detected from glial fibrillary associated protein (GFAP)-expressing astrocytes in cultures. Acute slices showed a burst frequency of 10.9 ± 4.2 bursts/min, which was not different from brainstem slice cultures (13.7 ± 10.6 bursts/min). However, slice co-cultures that include two cerebellar explants placed along the dorsolateral border of the brainstem displayed up to 193% faster burst frequency (22.4 ± 8.3 bursts/min) and higher signal amplitude (340%) compared to acute slices. We conclude that preBötC-containing slice cultures retain inspiratory-like rhythmic function and therefore may facilitate lines of experimentation that involve extended incubation (e.g., genetic transfection or chronic drug exposure) while simultaneously being amenable to imaging and electrophysiology at cellular, synaptic, and network levels.