(69b) High-Throughput Culture and Screening System for Microliter Droplets in CHO Cell Line Development

Chinese Hamster Ovary (CHO) cells have become the main cell chassis in the biopharmaceutical industry due to their human-like post-translational modifications, less secreted endogenous proteins, and ease of scalability. For the purpose of obtaining monoclonal cell lines which are stable, high-expression, and suitable for industrial cultivation in a short time, establishing a high-efficient cultivation and screening platform is a key technology in the cell line development (CLD). However, traditional method based on flasks and well plates have several disadvantages such as high labor costs, time-consuming, and low-screening efficiency. While automation can reduce manual labor, it still has limitations of high reagent and instrument costs. Therefore, a cost-effective, high-throughput screening platform is vital for advancing biotechnological development.

To increase the throughput of cell screening and characterization, recent technologies primarily utilize fluorescence intensity to identify high-expression cell lines. This method correlates fluorescent signal and cell expression capability, enabling the initial screening and collection of high-expression monoclonal cells into well plates. However, current fluorescence-based technologies are mainly focused on cell expression and require additional flasks or deep well plates screening to identify cells for industrial production, which raises downstream expenses and extends timelines. Consequently, a novel single cell microliter droplet screening system (MISS Cyto) that can screen cells according to secretory capability and growth performance simultaneously is established for CHO CLD. MISS Cyto is mainly composed of four modules: sampling module, droplet generation and cultivation module, micro-injection module, droplet detection and collection module. Firstly, we focused on the research of the microliter droplet generation and cultivation. Results showed that the generation of microliter droplet did not have obvious shear damage to cells, and the cell growth performance in microliter droplets was 1.3 times higher than that in well plates. We further generated single-cell microliter droplets based on Poisson distribution with a droplet generation rate of 7.0-7.5 Hz. It is proved that while MISS Cyto generating single-cell droplets quickly, it also achieves good monoclonality. Moreover, microliter droplets support the generation and cultivation of multi-cell droplets, offering an alternative to traditional well plate cell selection. In order to screen CHO cells with high target protein secretory capability in microliter droplet directly, GFP binding protein-1 (GBP-1) was selected as a fluorescent marker. After cultivation, the fluorescent probe is added via the micro-injection module. We constructed a CHO stable cell line co-expressing GBP-1 and target protein. By the droplet detection module, it is confirmed that GBP-fluorescent sensor shows high specificity and there was a linear correlation between signal intensity and secretory capability. Compared with other high-throughput screening methods, MISS Cyto greatly improves the throughput of CLD and simplifies the subsequent growth kinetics assessment. Given the high flexibility of MISS Cyto, it can be applied to gene or metabolic research of mammalian cells, single cell isolation and cultivation and other potential applications in the future.