Microscale controlled continuous cell culture

Abstract

Measurements of metabolic and cellular activity through substrate and product interactions are highly dependent on environmental conditions and cellular metabolic state. For such experiments to be feasible, continuous cultures are utilized to ensure consistent conditions. However, since medium must be replenished every cell doubling time, costs can be prohibitive in large reactors. An integrated microscale bioreactor with built-in fluid metering and environmental control will enable programmed experiments capable of generating reproducible data routinely. This work develops an instrument capable of supporting automated microscale continuous culture experiments. The instrument consists of a plastic-PDMS device capable of continuous flow reactions without volume drift. A novel bonding process is invented to fabricate devices with chemically stable interfaces against water, acids, and bases. We introduce a direct CNC machining and chemical bonding fabrication process for production of fluidic devices with a 1 mL working volume, high oxygen transfer rate (kLa ~ 0.025 s-1), fast mixing (2 s), accurate flow control (± 18 nL), and closed loop control over temperature, cell density, oxygen, and pH. Providing control over environmental parameters allows the system to perform different types of cell culture on a single device, such as batch, fed-batch, chemostat, and turbidostat continuous culture. Validation experiments demonstrate that cells can be grown to high optical densities (OD = 50) and production of commercially relevant chemicals such as DNA vaccines are comparable to large scale bench fermentations. Continuous cultures are also demonstrated without contamination for 3 weeks in a single device and both steady state and dynamically controlled conditions are possible, allowing observations of cell metabolic dynamics.