(52a) Continuous MINI-Reactors for CELL Culture Applications: Examples of Biopharmaceutical and Biomedical Applications

Mini-bioreactors have diverse applications in the field of biotechnology: screening of culture conditions, kinetic characterization of clones, and fundamental research in cell biology, among others. In this contribution, we illustrate the use of continuous mini-bioreactors for the culture of mammalian and yeast cells. In particular, we explore experimental scenarios relevant to biopharmaceutical and biomedical applications.  

We culture two recombinant strains in a continuous mini-device system of 2 mL of effective volume: (a) a recombinant Chinese Hamster Ovary (CHO) cell clone, producer of a monoclonal antibody (mAb), and (b) a recombinant strain of Pichia pastoris, producer of a viral antigen.  This mini-device, 3D printed in poly-lactic acid, was designed to function as 3-D continuous stirred-tank-type perfusion bioreactor were the residence time of the cells is greater than the mean hydraulic residence time due to the nature of the flow field induced by the device geometry.  In this system, mAb outlet steady state concentrations of 20-25 mg/L were observed in residence time of 16 h. MAb productivities exceed those exhibited by fully instrumented fed-batch stirred tank bioreactors of 1.5 L of (effective final volume). In preliminary experiments with P. pastoris cultures induced with methanol, outlet steady state concentrations of 50-100 µg/L of viral antigens were produced continuously.   

In addition, we use this continuous perfusion mini-reactor configuration for the culture and stem cell enrichment of different prostate cancer cells lines, namely PC3, 22RV1, DU145, and LnCaP cells. For all these cell lines, cells grow forming well developed spheroids with a cancer stem cell (CSC) content higher than 10% by continuously feeding and removing culture media at low flow rates (1-5 µL/min). The rate of growth of CSC enriched spheroids can be controlled by the hydraulic residence time.

We also present proof-of-principle results of the use of a different continuous mini-reactor configuration (1mL scale) for the production of monoclonal antibodies in adherent CHO cell cultures using zein-foams as the scaffold for cell adhesion. In these systems, monoclonal antibody concentrations of 60 mg/L were achieved under steady state conditions at residence times of 1 hr. The maximum cell density of CHO cell cultures grown in the foam mini-bioreactors exceed 10 fold that observed in their stirred tank counterparts. Remarkably, their productivity is 100 fold higher with respect to the one observed in optimized fed-batch cultures in instrumented bioreactors.

 Our results demonstrate the use of simple continuous flow mini-systems for cell culture applications pertinent to biopharmaceutical and biomedical applications.