Dataset Information

Continuous human iPSC-macrophage mass production by suspension culture in stirred tank bioreactors.

ABSTRACT: Macrophages derived from human induced pluripotent stem cells (iPSCs) have the potential to enable the development of cell-based therapies for numerous disease conditions. We here provide a detailed protocol for the mass production of iPSC-derived macrophages (iPSC-Mac) in scalable suspension culture on an orbital shaker or in stirred-tank bioreactors (STBRs). This strategy is straightforward, robust and characterized by the differentiation of primed iPSC aggregates into 'myeloid-cell-forming-complex' intermediates by means of a minimal cytokine cocktail. In contrast to the 'batch-like differentiation approaches' established for other iPSC-derived lineages, myeloid-cell-forming-complex-intermediates are stably maintained in suspension culture and continuously generate functional and highly pure iPSC-Mac. Employing a culture volume of 120 ml in the STBR platform, ~1-4 × 10⁷ iPSC-Mac can be harvested at weekly intervals for several months. The STBR technology allows for real-time monitoring of crucial process parameters such as biomass, pH, dissolved oxygen, and nutrition levels; the system also promotes systematic process development, optimization and linear upscaling. The process duration, from the expansion of iPSC until the first iPSC-Mac harvest, is 28 d. Successful application of the protocol requires expertise in pluripotent stem cell culture, differentiation and analytical methods, such as flow cytometry. Fundamental know-how in biotechnology is also advantageous to run the process in the STBR platform. The continuous, scalable production of well-defined iPSC-Mac populations is highly relevant to various fields, ranging from developmental biology, immunology and cell therapies to industrial applications for drug safety and discovery.

SUBMITTER: Ackermann M

PROVIDER: S-EPMC7612500 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Future industrial demand for large quantities of bacteriophages e.g., for phage therapy, necessitates the development of scalable Good Manufacturing Practice compliant (cGMP) production platforms. The continuous production of high titres of E coli T3 phages (1011 PFU mL-1) was achieved using two continuous stirred tank bioreactors connected in series, and a third bioreactor was used as a final holding tank operated in semi-batch mode to finish the infection process. The first bioreactor allowed the steady-state propagation of host bacteria using a fully synthetic medium with glucose as the limiting substrate. Host bacterial growth was decoupled from the phage production reactor downstream of it to suppress the production of phage-resistant mutants, thereby allowing stable operation over a period of several days. The novelty of this process is that the manipulation of the host reactor dilution rates (range 0.1⁻0.6 hr-1) allows control over the physiological state of the bacterial population. This results in bacteria with considerably higher intracellular phage production capability whilst operating at high dilution rates yielding significantly higher overall phage process productivity. Using a pilot-scale chemostat system allowed optimisation of the upstream phage amplification conditions conducive for high intracellular phage production in the host bacteria. The effect of the host reactor dilution rates on the phage burst size, lag time, and adsorption rate were evaluated. The host bacterium physiology was found to influence phage burst size, thereby affecting the productivity of the overall process. Mathematical modelling of the dynamics of the process allowed parameter sensitivity evaluation and provided valuable insights into the factors affecting the phage production process. The approach presented here may be used at an industrial scale to significantly improve process control, increase productivity via process intensification, and reduce process manufacturing costs through process footprint reduction.

Project description:BackgroundThe fungal natural products illudin S and M have been investigated as precursors for the development of semisynthetic anticancer agents such as Irofulven (illudin S derivative) which is currently in phase II clinical trials. Recently, illudin M derivatives have shown improved in vitro selectivity towards cancer cells encouraging further investigation. This requires a stable supply of the precursor which is produced by Basidiomycota of the genus Omphalotus. We have recently reported a robust shake flask process for the production of gram quantities of illudin M from Omphalotus nidiformis aiming to transfer that process into stirred tank bioreactors, which can be used in a commercial production set-up. However, process transfer across different systems is not straightforward and particularly challenging when the producer is morphologically complex. There are only a few reports that address the development of bioprocesses for the production of compounds from Basidiomycota as these organisms have not been extensively studied because of their complex life cycles and often are difficult to cultivate under laboratory conditions.ResultsThe recently developed shake flask process delivering stable titers of ~ 940 mg L-1 of illudin M was investigated using off-gas analysis to identify critical parameters which facilitated the transfer from shaken into stirred tank bioreactors. Comparable titers to the shake flask process were achieved in 2 L stirred tank bioreactors (1.5 L working volume) by controlling growth of biomass with a carefully timed pH-shift combined with an improved precursor-feeding strategy. A scale-up experiment in a 15 L bioreactor (10 L working volume), resembling the process at 1.5 L resulted in 523 mg L-1 and is the starting point for optimization of the identified parameters at that scale.ConclusionBy identifying and controlling key process parameters, the production process for illudin M was transferred from shake flasks into 2 L stirred tank bioreactors reaching a comparable titer (> 900 mg L-1), which is significantly higher than any previously reported. The insights obtained from 10 L scale pave the way towards further scale-up studies that will enable a sustainable supply of illudin M to support preclinical and clinical development programs.

Dataset Information

Continuous human iPSC-macrophage mass production by suspension culture in stirred tank bioreactors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets