Poster BB4 - Automated Temperature Control System for Circadian Entrainment in Vitro

Introduction: Although circadian rhythms control most physiological functions, few in vitro studies use circadian synchronized cultures as current synchronization methods are laborious and error-prone over the long term. For example, in vitro wound healing, tissue engineering, and lab-on-a-chip studies currently lack circadian synchronization, despite the widely recognized importance of circadian rhythms in these processes in vivo . To overcome the practical challenges of synchronizing cultures to a circadian rhythm, we developed Bioclockbot, a low-cost, open-source, automated temperature control system. Bioclockbot enables assignment of specific temperature profiles to individual culture dishes within an incubator, autonomously maintaining these temperatures over extended periods. This allows programming of periodic thermal stimuli known to effectively entrain circadian rhythms in various mammalian cell lines and tissues. In this work, we rigorously evaluated (1) the performance of the Bioclockbot's temperature control system against a state-of-the-art programmable incubator and (2) the entrainment of various cell lines (e.g., U2OS, fibroblasts, and mouse primary neurons) to periodic thermal stimuli provided by the Bioclockbot by examining circadian gene expression oscillations and rhythmicity in cellular functions. The validated Bioclockbot will be open source to offer the biomedical research community a more accessible way of developing in vitro physiological models that closely replicate their in vivo counterparts for biomedical research, without having to rely on chemical stimuli. This tool has broad applications for advancing our understanding of circadian rhythms across various fields.

Materials and

Methods: The Bioclockbot is an Arduino-based system that couples PT100 RTD temperature sensors and electrical heating pads using a proportional-integral-derivative (PID) controller. We designed a printed circuit board capable of independently controlling up to 5 well plates in parallel (Fig. 1A). Functional evaluation of the system included assessing its temperature regulation of culture dishes versus a state-of-the-art programmable incubator. To evaluate the system’s performance as a circadian entrainment tool, U2OS cells transfected with Per2: Luc or Bmal1: Luc reporters – which are key circadian gene reporters – were entrained with our system. We cycled the culture between 36 °C and 38.5°C every twelve hours for 72 hours based on previous protocols [1]. Bioluminescence readings (Lumicycle) were collected for 7 days and fitted to a dampening cosine curve. The output parameters are compared to temperature cycling entrainment realized by an incubator and synchronization with dexamethasone pulse serving as a positive control. Statistical analysis was performed with one-way ANOVA. In 2D wound scratch assays, entrained fibroblasts were scratched at different circadian times and the healings were measured and compared with non-entrained controls. To demonstrate the versatility of the device, an alternative thermal profile was applied to synchronize primary mouse neuron cultures that were extracted from day 0 C57BL/6N mouse pup hippocampus and cortex. These cultures were heat pulsed at 42°C for 15 mins and returned to 37°C for the remainder of the 24h cycle. Gene expression oscillations of Rev-Erb-α and Per2 (clock genes) were analyzed by RT-qPCR.
[1]Shin Yamazaki et al.,Science, 2000

Results, Conclusions, and Discussions: Our system reaches target temperatures, in both heating and cooling, significantly faster compared to a state-of-the-art incubator (Fig. 1B). Bioluminescence recordings of Bmal1 and Per2 expressions in U2OS cells reveal that temperature entrainment using our system induces circadian rhythms and yields the same period as the dexamethasone treatment and non-entrained cells (Fig. 1C). The system elicits rhythmic amplitudes significantly higher than incubator-programmed entrainment (Fig. 1C). Our system also demonstrates that precise temperature cycling can serve as a potent circadian entrainment cue in vitro, comparable to widely used chemical stimuli. Next, fibroblasts – which play key roles in wound healing – were transfected with Per2: Luc and the Per2 expression following Bioclockbot entrainment were recorded. Ongoing studies are testing these entrained cells in 2D and 3D in in vitro wound healing assays to investigate the circadian rhythmicity in cellular response to wounds. Additionally, heat pulse treatment (which involves a short spike in temperature) has been successfully demonstrated with this device, and the application of Bioclockbot in primary neuron cultures synchronization further supports the potentials of Bioclockbot being a powerful tool in a wide variety of areas of circadian research. The tool is easily fabricated, readily programmed and adjusted, and low cost.

Acknowledgements (Optional): This work is funded by NIH 5R35 Gm147272 grant.