A Scalable Cell Culture Perfusion System

DESCRIPTION (provided by applicant): Highly-integrated and complex tissue and organ culture-based assays, such as organotypic brain slices and dissociated cortical cultures, have great potential to contribute to the understanding of basic CNS processes, disease states, pharmacological and toxicological properties of drugs. The information-dense analyses of these cultures using multiunit extra cellular recordings from microelectrode arrays (MEA) or automated confocal imaging can provide information on neuronal connectivity and integration, firing patterns, and neuronal secretion in normal, drug-challenged and disease models. Building throughput into highly complex culture and assays systems such as MEAs will facilitate their wider application discovery, but will require sophisticated environmental control to limit variability and increase the quality and accuracy of these intricate analyses. Our alternative to expensive, custom designed integrated culture systems is to provide low cost, scalable fluidics for perfusion culture using hydraulic/pneumatic technology and computer control. ALA Scientific Instruments has developed controllable and highly accurate multi channel fluidic systems with this technology including Octaflow(tm), which can currently integrate up to 32 fluid channels. The system we envision for Phase I will integrate into investigator-optimized culture platforms. We will develop and test and refine core fluidic technology based on hydraulic/pneumatic and computer control with sensors to create an accurate system for sterile applications. A novel fluid sensor will be employed feedback control. The design will be tested on the bench top for reliability, consistency, and ability to maintain pH, CO2 and O2 levels by our academic collaborator who will also test the system for long-term sterility in mock MEA cultures and for ability to maintain and even experimentally manipulate MEA cultures of organotypic suprachiasmatic nuclear explant from mouse. In Phase II our goal will be to design and test a scaled-up unit, which will include expanded sensor capabilities and adaptors for application to a larger number of commercially available culture vessels. Small chambers that hold 1-3ml of solution are more and more frequently being used to store and grow live cell cultures. Healthy tissue growth requires the exchange of fluid media and the availability of oxygen. Metabolic waste must be removed and nutrients need to be brought in. In the case of long term cultures where electrical or optical data recording takes place from the living tissue be it neurons, heart tissue or kidney, etc. long term stability, and sterility are a must. Small peristaltic pumps have been shown to do a very good job at exchanging media in small chambers, and two pumps can do a very good job on a single small chamber. However, the rise of high-throughput systems which require multiple identical platforms of cultured tissue mean that pump based systems would require a lot of pumps. This is problematic because for example a 96 well plate would need 192 pumps to provide each well with an input and output tube. These pumps often cost $500 each, and the power requirement and foot print for so many would make such a system prohibitive. (Not to mention all the moving parts and maintenance etc.) To solve the multiplexing problem we believe we can substitute valves for pumps. Valves are often used in sterile environments, many are FDA approved, and their cost (about 10% of peristaltic pumps), energy requirements, and footprint lend them to this type of application. All that is required is a pneumatic drive system to provide pressure and suction. We propose to test the feasibility of such a valve based system in this phase one project. In addition, we will supplement our system with drop counters and level sensors we have already worked on, to make the systems accurate and dependable.