Robust bioprocess design and evaluation of commercial media for the serial expansion of human induced pluripotent stem cell aggregate cultures in vertical-wheel bioreactors

Abstract

Background: While pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified. Methods: This study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture. Results: The robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth

1. Introduction

One of the most urgent problems in regenerative medicine is a lack of suitable source of cells, tissues and organs used to replace or repair the biological function of damaged tissues. Pluripotent stem cells (PSCs) have generated significant attention owing to their capacity for self-renewal and ability to differentiate into all three germ layers. Once established, human PSC (hPSC) lines have almost unlimited proliferation capacity and can retain the ability to give rise to all cell lineages, making them an ideal platform material for cell-based therapies.

Successful implementation of a PSC-based therapy for clinical and commercial purposes will rely on the development of a robust and scalable cell culture process for the expansion and differentiation of these cells to enable production of a desired target number of specific cells (i.e., a manufacturing lot size to meet the clinical or commercial needs) in a consistent manner. PSCs are traditionally grown in static culture vessels as adherent monolayers or non-adherent spherical aggregates. Although sufficient to generate cells for experimental purposes, this approach is impractical to achieve large quantities required for clinical or commercial applications. For PSC-based treatments, cell dosages will range from 109 to 1012 cells per patient depending on the therapeutic target [1]. To achieve the required number of cells in an effective manner, scalable bioreactors will need to be used. Biomanufacturing of cells for therapeutic purposes using such bioreactors is advantageous due to reduced operating and labor cost requirements, improved scalability, and the ability to fine-tune process control capabilities [2].

Suspension bioreactors have been employed by the bioprocessing industries for decades for mass production of recombinant proteins and monoclonal antibodies using well-established cell lines (such as Chinese Hamster Ovary cells) that grow well in dynamic liquid-mixing culture environments. However, suspension cultures introduce hydrodynamic forces which have been shown to impact various stem cell attributes such as proliferation