The development of Integrated Bioprocesses is a crucial demand to overcome the main technological barriers that presently limit the application of human Pluripotent Stem Cells (PSC), embryonic (ESC) and induced pluripotent (iPSC), and their derivatives in Regenerative and Precision Medicine. This progress is expected to leverage the emergence of alternative therapies and the development of new personalized tissue models for disease modelling and drug discovery. Bioprocessing approaches that are being developed at SCERG include:
- Scalable expansion of hiPSC while maintaining their pluripotency. In particular, xeno-free culture systems are being explored as a means to improve the reproducibility and robustness of the bioprocess and to facilitate further translation of hiPSC-derived products into clinical applications [1, 2];
- Scalable integrated expansion and controlled neural and cardiac differentiation of hiPSCs by culturing these cells as 3D aggregates in suspension [3, 4]. This culture platform is being used namely for production of neural precursors under chemically-defined conditions, by performing the spatial and temporal control of cell aggregation [3, 4];
- Downstream processing methodologies for hiPSC and their differentiated derivatives and their integration with the scalable expansion and differentiation of hiPSC. Separation of hiPSC from microcarriers after expansion is performed namely using dissolvable microcarriers. Cell separation methods based on affinity principles are being focused for purification of hiPSC derivatives after the process of differentiation [4]. This includes the depletion of tumorigenic hPSC that remain in the bioprocess after neural differentiation [5];
- Development of standardized culture platforms for production of neural and cardiac tissues from healthy and patient-specific hiPSCs for disease modelling and drug screening. A chemically-defined monolayer-based culture system for neural differentiation of patient-specific hiPSC is being explored for modelling of Rett Syndrome [6]. Size-controlled aggregates of hiPSC-derived neural precursors are being produced in microwells for high-throughput neurotoxicology assays.
Selected publications:
[1] Badenes, S.M., Fernandes, T.G., Cordeiro, C.S.M., Boucher, S., Kuninger, D., Vemuri, M.C., Diogo, M.M., Cabral, J.M.S. Defined essential 8™ medium and vitronectin efficiently support scalable xeno-free expansion of human induced pluripotent stem cells in stirred microcarrier culture systems. PLoS One 11:e0151264 (2016).
[2] Badenes, S.M., Fernandes, T.G., Miranda, C.C., Pusch-Klein, A., Haupt, S., Rodrigues, C.A.V., Diogo, M.M., Brüstle, O., Cabral, J.M.S. Long-term expansion of human induced pluripotent stem cells in a microcarrier-based dynamic system. J Chem Technol Biotechnol 92:492-503 (2017).
[3] Miranda, C.M., Fernandes, T.G., Pascoal, J.F., Haupt, S., Brüstle, O., Cabral, J.M.S., Diogo, M.M. Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment. Biotechnol J 10(10):1612-1624 (2015).
[4] Miranda, C.C., Fernandes, T.G., Diogo, M.M., Cabral, J.M.S. Scaling Up a Chemically-Defined Aggregate-Based Suspension Culture System for Neural Commitment of Human Pluripotent Stem Cells. Biotechnol J 11(12):1628-1638 (2016).
[5] Rodrigues, G.M.C., Matos, A.F.S., Fernandes, T.G., Rodrigues, C.A.V., Peitz, M., Haupt, S., Diogo, M.M., Brüstle, O., Cabral, J.M.S. Integrated Platform for Production and Purification of Human Pluripotent Stem Cell-derived Neural Precursors. Stem Cell Rev Rep 10(2):151-161 (2014).
[6] Fernandes, T.G. Engineering at the microscale: A step towards single-cell analysis of human pluripotent stem cells. Biotechnol J 10(10):1511-1512 (2015).