Evaluation of Perfusion Cell Culture Conditions in a Double-Layered Microphysiological System Using AI-Based Morphological Analysis

Background: In recent years, microphysiological systems (MPS) using microfluidic technology as a new in vitro experimental system have shown promise as an alternative to animal experiments in the development of drugs, especially in the field of drug discovery, and reports have been published that MPS experiments have replaced animal experiments. We have commercialized the Fluid3D-X®, a double-layer microfluidic chip made of polyethylene terephthalate (PET), under the Japan Agency for Medical Research and Development (AMED) MPS development research project and have applied it to various organ models. When Caco-2 cells were cultured using Fluid3D-X® and a peristaltic pump, villi-like structures were formed in the microchannels, but the degree of formation differed between the upstream and downstream sides. To examine consideration points regarding the effects of the nutrient and oxygen supply by the chip material and medium flow on cells in the widely used double-layer microfluidic chip, and demonstrate the usefulness of a new imaging evaluation method using artificial intelligence technology for the morphological evaluation of cells. Methods: The following studies were conducted. 1. Perfusion direction: The medium was perfused at a normal flow rate (20 µL/min) using Fluid3D-X® and a peristaltic pump in opposite directions within the top and bottom channels and in the same direction initially, then reversed in both layers. 2. Perfusion speed: Incubated at normal and doubled (40 µL/min) flow rates using Fluid3D-X® and a peristaltic pump. 3. Materials: Incubated at the normal flow rate with Fluid3D-X® and polydimethylsiloxane (PDMS) chip, which has the same microchannel design as Fluid3D-X® but is made of PDMS. Results: Cell morphology in the channels was quantified and evaluated using Nikon NIS.ai in addition to microscopic observation. Villi-like structures were predominant upstream of the top channel, independent of perfusion on the bottom channel, and those structures downstream increased with doubled speed. Additionally, compared to the Fluid3D-X, the chip made of PDMS showed almost uniform villi-like sterilization in the channel. Conclusion: As the amount of oxygen and nutrients required by different cell types differs, it is necessary to study the optimization of culture conditions according to the characteristics of the cells handled.