Human pluripotent stem cells (hPSCs) and their derivatives have gained increased importance for industrial applications in recent years. They have a great potential for therapeutic applications as well.
In vitro assays and novel regenerative therapies will require large cell quantities produced under defined conditions.
In conventional mammalian cell culture, the utilization of bioreactors is well established, e.g., for the production of recombinant therapeutic proteins, vaccines, and antibodies. Established protocols are used for process development and manufacturing of mammalian cells in 100–1,000-L scale and beyond.
Cell cultivation in stirred tank bioreactors allows for tight control and online monitoring of all relevant process parameters such as temperature, agitation, pH or dissolved oxygen, and scaleup.
This know-how can serve as a basis for creating processes to cover the demand on hPSCs, including human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs).
There already have been investigations on cultivating adherence-dependent stem cells in suspension culture. These studies mainly focused on the modification of matrix-attached hESC cultivation on microcarriers, a method that is widely used in conventional mammalian cell culture.
However, culture heterogeneity due to the preference of undifferentiated hPSCs to stick to each other rather than to prescreened types of microcarriers might be challenging. Recent studies in our labs and elsewhere have now demonstrated the potential of cultivating undiffentiated human ESCs and iPSCs as cell-only-aggregates in suspension.
More recently, we have demonstrated the feasibility of translating this approach into stirred tank reactors, paving the way for the envisioned mass production of pluripotent stem cells and their derivatives. Key features of this technology include utilization of a fully defined serum-free culture medium, single cell-based inoculation, and significant long-term expansion of hPSCs in easy-to-scaleup suspension independent of extracellular matrices or scaffolds.
The method was successfully applied to several human hPSC lines and cynomolgus monkey ES cells as well. In a previous step, transfer from static suspension in culture dishes to stirred spinner and shaken Erlenmeyer flasks was also enabled.
Such dynamic cultivation of cell-only-aggregates turned out to be robust regarding the reproducibility of cell expansion, karyotype stability, and overall preservation of the stem cells’ pluripotency. Somewhat lower expansion rates in dynamic conditions further suggested a high potential for culture optimization by applying a more controlled environment.
To improve culture monitoring and control capabilities and to pave the way for larger-scale cultivation, the method was transferred to a stirred tank bioreactor system.
Studies were carried out in a Dasgip parallel bioreactor system consisting of four 250 mL cultivation vessels (100–250 mL working volume) including an integrated Dasgip control unit and software. Allowance was made for independent monitoring and control of temperature, pH, oxygen tension, and stirring conditions.
To test and ensure reproducibility, all approaches were performed in four independent experimental repeats in a culture volume of 125 mL each. Utilizing cord blood endothelial cell-derived hiPSCs, preliminary experiments showed that the inoculation density and the agitation mode were highly critical for successful process initiation in impeller-stirred bioreactors.
Experiments revealed that 5x105 hPSCs/mL was an efficient inoculation density in the stirred bioreactor setting. Subsequently, experiments were carried out to compare impeller designs and stirring speed modulation aiming at optimal control of cell aggregation and homogeneity of aggregate size distribution.