Inertial-based Microcarrier-cell retention in bioprocessing

Abstract

Background & Aim Cell therapy has gained momentum in recent years. To meet the increasing demand for a large number of adherent cell types such as MSC transplantation, a paradigm shift is therefore inevitable from monolayer cell culture flask to highly dense 3D cell culture environment to achieve throughput of up to 100 billion to trillion cells per lot. One alternative approach is using microcarrier (∼100 to 300 µm size) as suspension scaffold in stirred tank bioreactors to scale up cell expansion. Despite prominent advantages of microcarrier-based bioreactors, this technology also surfaces some hurdles such as removal of particulates, for example, microcarrier fragments generated due to high shear rate in bioreactors and retention of microcarrier-cell complex for perfusion culture. Here we demonstrated for the first time retention of microcarrier-cell complex using scaled-up inertial microfluidic systems. Methods, Results & Conclusion Emerging inertial microfluidics technique as an alternative method to microfiltration enables continuous and clog-free separations with lower maintenance costs. In this study, we developed a large-scale inertial microfluidic system suitable for high-throughput microcarrier-cell retention from perfusion bioreactors. Microcarrier-cell complex as a result of the interplay between shear-gradient lift force and secondary drag force start to migrate towards the outer wall as traveling inside a trapezoidal spiral channel (Fig. 1A-B). A series of experiment was carried out and the optimized single-loop trapezoidal spiral channel was found. Figure 1C shows bright-field images of Cytodex-3 microcarriers (GE Healthcare) equilibrated at the outer half of channel cross-section before bifurcation and separated from the outer wall outlet. The best focusing with minimum particle band width was developed at a flow rate of 16 mL/min (Re≈200). This optimized flow condition can deal with suspensions having high microcarrier volume fraction as much as ∼3.4 % to filter microcarriers from the outer wall (OW) outlet (Fig. 1D). As a proof-of-concept demonstration, a case model was prepared with Cytodex-3 MCs (∼2 % volume fraction) and hMSCs suspension (∼2.4 × 104 cells/mL). During 7-day culture, the single-loop trapezoidal channel performed medium exchange at day 2, 4 and 6 and inlet flow rate of 20 mL/min. The proposed method is validated by assessing the immunophenotypic expression of harvested MSCs at day 7 according to ISCT criteria (Fig. 1E).