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novel bioreactor Platform for reprOducible, Scalable ExpansIon and cardiac DifferentiatiON of hiPSC in suspension culture

Periodic Reporting for period 1 - POSEIDON (novel bioreactor Platform for reprOducible, Scalable ExpansIonand cardiac DifferentiatiON of hiPSC in suspension culture)

Reporting period: 2015-10-01 to 2017-09-30

Human induced pluripotent stem cells (hiPSCs) have the potential to revolutionize biomedical sciences, however their standardised mass production, indispensable for industrial and therapeutic use, is still a major challenge. Impeller stirred-tank bioreactors, currently used for hiPSC bioprocessing, impose detrimental stresses on cultured hiPSCs, resulting in heterogeneity of cells and cell-aggregates, limited bioprocess reproducibility, and low hiPSC expansion efficiency at high costs. The main purpose of the POSEIDON project was the development of a next-generation automated bioreactor fully compatible with good laboratory and manufacturing practise (GLP/GMP) standards, and suitable to improve hiPSC bioprocessing.
Starting from an existing proof-of-concept prototype, the bioreactor has been developed and optimized for hiPSC suspension culture under non-impeller-dependent low-shear hydrodynamic conditions. The functioning principle of the bioreactor is based on the recirculation of the culture medium inside the culture chamber under laminar flow regime, obtained through the modulation of the perfusion system flow rate, in order to produce low shear stress dynamic suspension conditions. Due to the peculiar geometric features of the culture chamber, such a system promotes the establishment of buoyant vortices that maintain cells/constructs in dynamic suspension, minimizing their sedimentation, and in parallel it avoids the use of impellers and/or rotational components, that could induce detrimental stresses on cells. Developed by advanced engineering design methods, the bioreactor has been equipped with novel automated systems (Fig.1) based on smart and cost-effective technological solutions to guarantee: 1) continuous medium perfusion and renewal, to maintain homogeneous culture conditions and preserving the hiPSC-released factors; 2) online monitoring of culture parameters and adaptive control of processing conditions; 3) control of cell aggregate size distribution through the pump operating mode. A computational multiphysics approach has been adopted for identifying the optimal operating conditions for hiPSCs dynamic suspension culture.
An additional and unexpected outcome of the project has been the discovery that peristaltic pump-based approaches can provoke insulin precipitation in the perfused culture media, consequently inducing hiPSC viability loss. This finding has substantial impact within and beyond hPSC manufacturing in the perspective of developing chemically defined culture media and establishing novel strategies for automated expansion of hPSCs.
A detailed description of the work performed during the project and the related scientific publications can be found in the Technical Report.

The POSEIDON achievements have been disseminated through high quality scientific publications, participating to different international conferences and workshops focused on stem cell research and manufacturing technology for stem cell bioprocessing, and presenting periodic progress reports at the MHH. This dissemination plan allowed to present the project results to research experts and to the European stem cell manufacturing, bioreactor and pharmaceutical industries, supporting the Fellowship impact on European excellence and competitiveness.

List of publications with POSEIDON result dissemination:
1. Massai D, Bolesani E, Diaz D, Kropp C, Kempf H, Halloin C, Martin U, Braniste T, Isu G, Harms V, Morbiducci U, Dräger G, Zweigerdt R. Sensitivity of human pluripotent stem cells to insulin precipitation induced by peristaltic pump-based medium circulation: Considerations on process development. Scientific Reports Nature Publishing Group,7:3950, 2017, open access
2. Kropp C, Massai D, Zweigerdt R. Progress and challenges in large-scale expansion of human pluripotent stem cells. Process Biochemistry Elsevier Ltd, 244-254, 2017, open access
3. Chimenti I, Massai D, Morbiducci U, Beltrami AP, Pesce M, Messina E. Stem Cell Spheroids and Ex Vivo Niche Modeling: Rationalization and Scaling-Up. Journal of Cardiovascular Translational Research Springer New York LLC, 150-166, 2016

List of attended international conferences with POSEIDON result dissemination:
1. 4th International Annual Conference of the German Stem Cell Network (GSCN), 12 - 14 September 2016, Hannover (Germany). Poster presentation “Severe reduction of hPSC vitality is mediated by insulin stability in culture media: impact of physicochemical conditions on bioprocess development”
2. 1st Stem Cell Community Day - Bioprocessing Technologies in Stem Cell Research: Challenges and Chances for Commercializiation, 04 April 2017, Hamburg (Germany). Poster presentation “Insulin precipitation induced by peristaltic pump-based medium circulation impairs culture of human pluripotent stem cells”. Poster Award 1st place
3. ISSCR 2017 – Annual Meeting of the International Society for Stem Cell Research, 14 – 17 June 2017, Boston (United States). Poster presentation “Sensitivity of human pluripotent stem cells to insulin precipitation induced by peristaltic pump-based culture medium circulation”

In the perspective of establishing novel strategies for automated propagation of hPSCs, the development of the bioreactor and the discovered physical instability of insulin induced by peristaltic pump-based approaches open interesting scenarios within and beyond hPSC manufacturing.
The POSEIDON knowledge and outcomes can positively impact on the European biomedical, pharmaceutical, and cell therapy research and business sectors. In particular, the developed bioreactor represents an alternative solution for dynamic suspension culture crucial for culturing cells/organisms that need low-shear stress conditions. Moreover, the discovery that peristaltic pump-based approaches can provoke insulin precipitation and therefore inducing hiPSC viability loss is crucial in the perspective of developing chemically defined culture media and establishing novel strategies for automated expansion of hPSCs. This knowledge has substantial impact within and beyond hPSC manufacturing. This will contribute to the European “Innovation Union”, supporting crucial research and health sectors with consequent European excellence and competitiveness in the exponentially growing stem cell global market, with the final aim of improving quality of life in Europe.