Supplementary figures for "Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor"

Supplementary information files for "Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor"<div><br></div><div><p><b>Figure S1</b>. Growth kinetics of hMSCs donor 2 cells using serum-free (SFM) and fetal bovine serum (FBS)-based media in both the ambr15 and spinner flasks with data showing the viable cell density.</p><p><br></p><p><b>Figure S2</b>. Nutrient and metabolite flux for hMSC donor 1 cells expanded on microcarriers in the serum-based and serum-free cultures in both the ambr and spinner flasks.</p><p><br></p><p><b>Figure S3</b>. Functional characterisation of hMSCs from donor 1 harvested from the serum-free ambr15 bioprocess.</p><p><br></p><h2>ABSTRACT</h2><p>Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, N<sub>JS</sub>. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency.</p></div>