Three-dimensional cell-based high-throughput screening for drug discovery and cell culture process development.

摘要

High-throughput screening (HTS) is widely used in the pharmaceutical industry for target identification and hit/lead selection. However, conventional HTS often generates a large number of poorly qualified leads that must go through expensive and time-consuming animal experiments. The development of a new HTS platform with more relevant in-vivo biological information that can reduce the number of leads into animal experiments is thus critical to the drug discovery process. Based on three-dimensional (3-D) cultures of GFP-expressing mammalian cells, a novel microbiorector array capable of online monitoring of biological activities was developed for high-throughput drug screening. The 3-D microbioreactor array can afford parallel, automated, and long-term (over one month) cell bioactivity assays. It can also increase signal to noise ratio (SNR) by at least one order of magnitude as compared to the conventional 2-D culture system and at the same time remove most of the detection interference caused by cell activities. It used inexpensive materials and proven tissue engineering principles, and can be used for fast cell culture media development and cytotoxicity assays for drug screening.; Toxicity of embryotoxic reference chemicals and anti-cancer drugs was measured in the 3-D multicellular models and the predicted toxicity was compared to that from monolayer cultures. It showed that the 3-D system was a more realistic pharmacotoxicological test system than 2-D monolayer cultures. With the 3-D system, acquired tissue resistance in the treatment of bulky tumor tissues could be revealed in a high-throughput manner. As a bridge over the gap between monolayer cell cultures and animal models, this 3-D system can improve the drug discovery process when being applied in toxicity and efficacy tests prior to animal experiments.; Besides high-throughput toxicity screening, autofluorescence detection of 3-D tissue cultures could also be extended to immobilized cell culture process development. Butyrate treatment was used as a case study to demonstrate the performance of the new system. The microbioreactor array developed was used for high-throughput cell process development to improve monoclonal antibody (MAb) production in a fibrous bed bioreactor (FBB) using CHO cells. A novel online fluorescence probe was developed and used in spinner flasks and a lab-scale perfusion fibrous bed bioreactor to non-invasively quantify cell growth and MAb productivity. The results from this study showed that GFP fluorescence could indicate recombinant protein production and thus provide a fast, reliable and robust platform for cell culture process development to optimize target protein productivity without cell counting or protein analysis.