A two-step microengineered system for high-density cell retention from bioreactors

Abstract

Large-scale cell culture processes are required to produce biopharmaceuticals, cells for tissue engineering, and vaccine production while being effective in toxicity testing, gene therapy vector production for cancer research, and drug development. A growing trend in these industries, particularly for suspension cells, involves implementation of continuous cell perfusion processes, which require an aseptic, efficient, cost-effective, and reliable cell separation and retention scheme. Many cell separation techniques (membrane-based systems, lateral displacement devices, and acoustophoresis) have proven to be highly efficient, but suffer from issue of clogging and high cost, limiting their reliability, and thus, their overall feasibility. Some cell retention devices—those based on inertial microfluidics—offer high reliability (i.e., clog-free), but their efficiency reduces at higher cell concentrations. To overcome this apparent trade-off, we report the development of an integrated system consisting of two different membrane-less microfiltration techniques for cell separation from spent cell media. Although it could be adapted to numerous cell culture applications, this system was optimized and tested for suspension-adapted Chinese Hamster Ovary (CHO) cells. As the first step of the cell retention system, a miniaturised hydrocyclone was developed that could separate the cells with macroscopic volume processing rates (~200 mL/min). At this stage, up to 75% of the cells were isolated with minimal (<5%) change in the viability. The remaining cells passed through the overflow of the device and entered to a multiplexed spiral microchannel system, where more than 90% of the remaining cells were recovered, yielding an overall efficiency of up to 95%. The proposed integrated system is thus ideal for continuous and high throughput cell retention even at high cell concentrations (~80 million cells/mL), which is in range of current need in the bioprocessing industry.