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"Figure S1. 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.Figure S2. 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.Figure S3. Functional characterisation of hMSCs from donor 1 harvested from the serum-free ambr15 bioprocess.ABSTRACTMicrobioreactors 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, NJS. 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.

补充信息文件：《利用自动化高通量微生物反应器开发人源多能骨髓间充质细胞微载体培养工艺的工艺开发》图S1. 在ambr15和旋涡瓶中，使用无血清（SFM）和胎牛血清（FBS）为基础的培养基培养hMSCs供体2细胞的生长动力学，数据展示了可存活细胞密度。图S2. 在血清和血清-free培养基中，hMSC供体1细胞在微载体上扩展的营养物和代谢物通量，实验在ambr和旋涡瓶中进行。图S3. 从血清-free ambr15生物工艺中收获的hMSCs供体1的功能特性。摘要：微生物反应器在工艺开发中发挥着至关重要的作用，它们能够降低试剂需求，并有助于提高通量筛选工艺参数和培养条件。在此，我们首次详细展示了自动化ambr15细胞培养微生物反应器系统在开发可扩展的人源间充质多能骨髓/干细胞（hMSC）微载体培养工艺中的适用性。这一成就通过首先优化微载体的悬浮和混合，然后改善细胞附着，从而缩短了初始生长滞后期。后者是通过在最初的24小时内仅使用培养基最终工作体积的50%，并采用间歇搅拌策略实现的。这些变化导致与原始工艺（24小时不搅拌，100%工作体积）相比，24小时后的可存活细胞密度增加了超过150%。使用与ambr15相同的方法，在更大规模的旋涡瓶研究中也获得了类似改进。最后，基于血清型培养基的改进生物工艺方法被应用于ambr15的血清-free工艺中，与血清型工艺相比，产量提高了超过250%。在两个规模中，培养过程中使用的搅拌是微载体悬浮所必需的最小量，NJS。与旋涡瓶相比，ambr15的改进控制减少了含血清培养基中可存活细胞密度的变异系数，从7.65%降至4.08%，而转向血清-free进一步将这些降低至1.06–0.54%。血清-free和自动化处理的结合，与血清型、手动旋涡瓶工艺相比，提高了超过10倍的再现性。本研究的结果表明，ambr15微生物反应器是hMSC微载体培养生物工艺开发的有效工具，而血清-free培养基、控制和自动化的结合，则提高了工艺产量和一致性。