Stem cell bioprocessing and cell therapy

Stem cells (SCs), with their ability for extensive proliferation and multi-lineage differentiation, can serve as a renewable source of cellular material in regenerative medicine and toxicology. A pre-requisite for the transition of SCs or their progeny to these fields is the establishment of efficient cell culture protocols for large-scale expansion, differentiation, storage and distribution. This efficiency will be measured in terms of robustness, reproducibility, scalability and automation, as well as compatibility with ATMP (Advanced Therapy Medicinal Products) regulations from EMA, and compliant with "Good Manufacturing Practices” – GMP. The major challenges in producing SC-derivatives are the scaling up of reproducible pure cell populations of undifferentiated cells without compromising their self-renewal ability and differentiation potential and the directed differentiation to specific cell types with improved differentiation efficiency, high purity and cell functionality.

The potential for producing novel cell-based products from stem cells is large. We are applying our expertise in bioreactor technology to the development of effective and scalable culture systems that can integrate stem cell in vitro expansion, their target differentiation and cryopreservation. We have been using stirred-tank bioreactors, that allow process scale-up and automation, as well as efficient monitoring and control of the culture environment. For on-line and non-invasive phenotypic monitoring and selection, our group is developing new reporter hESC cell lines. Furthermore, by integrating new approaches for cryopreservation, it will be possible to distribute high quantities of ready-to use cells with improved viability and functionality. We are currently working with embryonic and adult stem cells, of human and murine origin.

Selected Publications

  1. Correia C, Koshkin A, Duarte P, Hu D, Carido M, Sebastião MJ, Gomes-Alves P, Elliott DA, Domian IJ, Teixeira AP, Alves PM, Serra M. 2018. 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes. Biotechnol Bioeng. 2018 Mar;115(3):630-644
  2. Bárbara Cunha, Ricardo J.S. Silva, Cláudia Correia, Alexey Koshkin, Paula M.Alves, Margarida Serra, Cristina Peixoto, Manuel J.T. Carrondo. 2017. Finding the design space of a filtration-based operation for the concentration of human pluripotent stem cells. Journal of Membrane Science 399-407.
  3. Correia C, Koshkin A, Duarte P, Hu D; Teixeira AP, Domian I, Serra M,  Alves PM. 2017Distinct carbon sources affect structural and functional maturation of human pluripotent stem cells derived cardiomyocytes. Sci Rep. Aug 17;7(1):8590
  4. Cunha B, Aguiar T, Carvalho SB, Silva MM, Gomes RA, Carrondo MJT, Gomes-Alves P, Peixoto C, Serra M, Alves PM. 2017. Bioprocess integration for human mesenchymal stem cells: from up to downstream processing scale-up to cell proteome characterization.  J Biotechnol. S0168-1656(17)30031-7.  
  5. Abecasis B, Aguiar T, Arnault E, Costa R, Gomes-Alves P, Aspegren A, Serra M, Alves PM. 2017. Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.  J Biotechnol. 246:81-93. 
  6. Sá JV, Kleiderman S, Brito C, Sonnewald U, Leist M, Teixeira AP, Alves PM.  2017.  Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis. Neurochemical Research. 42, 244-253  
  7. Gouveia PJ, Rosa S, Ricotti L, Abecasis B, Almeida HV, Monteiro L, Nunes J, Carvalho FS, Serra M, Luchkin S, Kholkin AL, Alves PM, Oliveira PJ, Carvalho R, Menciassi A, das Neves RP, Ferreira LS. 2017. Flexible nanofilms coated with aligned piezoelectric microfibers preserve the contractility of cardiomyocytes. Biomaterials. 139:213-228. 
  8. Gomes-Alves P, Serra M, Brito C, Ricardo CP, Cunha R, Sousa MF, Sanchez B, Bernad A, Carrondo MJT, Rodriguez-Borlado L, Alves PM. 2016. In vitro expansion of human cardiac progenitor cells: exploring 'omics tools for characterization of cell-based allogeneic products. Translational Research
  9. Cunha B, Silva RJ, Aguiar T, Serra M, Daicic J, Maloisel JL, Clachan J, Åkerblom A, Carrondo MJ, Peixoto C, Alves PM. 2016. Improving washing strategies of human mesenchymal stem cells using negative mode expanded bed chromatography. J Chromatogr A. 2016 Jan 15;1429:292-303. 
  10. Silva MM, Rodrigues AF, Correia C, Sousa MQF, Brito C, Coroadinha AS, Serra M, Alves PM. 2015. Robust expansion of human pluripotent stem cells: integration of bioprocess design with transcriptomic and metabolomic characterization. Cells and Translational Medicine, Jul;4(7):731-42 
  11. Cunha B, Aguiar T, Silva MM, Silva R, Sousa MF, Pineda E, Peixoto C, Carrondo MJ, Serra M, Alves PM. 2015. Exploring continuous and integrated strategies for the up- and downstream processing of human mesenchymal stem cells. Journal of Biotechnology - special issue on integrated continuous biomanufacturing, ahead of print 2015 Mar 4,   
  12. Suter-Dick L, Alves PM, Blaauboer BJ, Bremm KD, Brito C, Coecke S, Flick B, Fowler P, Hescheler J, Ingelman-Sundberg M, Jennings P, Kelm JM, Manou I, Mistry P, Moretto A, Roth A, Stedman D, van de Water B, Beilmann M. 2015. Stem Cell-Derived Systems in Toxicology Assessment. Stem Cells Dev. Mar 31. [Epub ahead of print]   
  13. Cunha B, Peixoto C, Silva MM, Carrondo MJT, Serra M, Alves PM, 2015
  14. Filtration methodologies for the clarification and concentration of human mesenchymal stem cells. J Memb Sci, Volume 478, 117-129. 
  15. Gomes-Alves, P., Serra, M., Brito, C., R-Borlado L, Lopez JA, Vazquez J, Carrondo MJT, Bernad A & Alves PM. 2015. Exploring analytical proteomics platforms toward the definition of human cardiac stem cells receptome. Proteomics 15, 1332-7
  16. Cunha B, Peixoto C, Silva MM, Carrondo MJT, Serra M, Alves PM. 2015. Filtration methodologies for the clarification and concentration of human mesenchymal stem cells. J Memb Sci, Volume 478, 117-129
  17. Simão D, Pinto C, Piersanti S, Weston A, Peddie CJ, Bastos AEP, Licursi V, Schwarz SC, Collison LM, Salinas S, Serra M, Teixeira AP, Saggio I, Lima PA, Kremer EJ, Schiavo G, Brito C, Alves PM. 2015. Modelling human neural functionality in vitro: 3D culture for dopaminergic differentiation. Tissue Engineering Part A 21, 654-668. 
  18. Correia C, Serra M, Espinha N, Sousa M, Brito C, Burkert K, Zheng Y, Hescheler J, Carrondo MJ, Sarić T, Alves PM. 2014. Combining Hypoxia and Bioreactor Hydrodynamics Boosts Induced Pluripotent Stem Cell Differentiation Towards Cardiomyocytes. Stem Cell Reviews and Reports. Published on line 15 July 2014. 
  19. Gualda EJ, Simão D, Pinto C, Alves PM, Brito C. 2014. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence. Frontiers in Cellular Neuroscience. Published on line 6 August 2014. 
  20. Malpique R, Tostões R, Beier AF, Serra M, Brito C, Schulz JC, Björquist P, Zimmermann H, Alves PM, 2012. Surface-based cryopreservation strategies for human embryonic stem cells: A comparative study. Biotechnol Prog, 28(4):1079-87. 
  21. Serra M, Brito C, Correia C, Alves PM, 2012. Process engineering of human pluripotent stem cells for clinical applications. Trends Biotechnol, 30(6):350-9. 
  22. Brito C, Simão D, Costa I, Malpique R, Pereira CI, Fernandes P, Serra M, Schwarz S, Schwarz J, Kremer EJ, Alves PM. 2012. 3D cultures of human neural progenitor cells: Dopaminergic differentiation and genetic modification, Methods 56, 452–460. 
  23. Serra M, Correia C, Malpique R, Brito C, Jensen J, Bjorquist P, Carrondo MJ, Alves PM, 2011. Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells. Plos One, 6(8) e23212. 
  24. Vieira HL, Alves PM, Vercelli A, 2011. Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species. Prog Neurobiol, 93:444-55. 
  25. Serra M, Brito C, Sousa MF, Jensen J, Tostões RM, Clemente JJ, Hyllner J, Strehl R, Carrondo MJ, Alves PM, 2010. Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control. J Biotechnol, 148(4):208-15. 
  26. Malpique R, Osório LM, Ferreira DS, Ehrhart F, Brito C, Zimmermann H, Alves PM, 2010. Alginate encapsulation as a novel strategy for the cryopreservation of neurospheres. Tissue Eng Part C Methods, 16(5):965-77. 
  27. Malpique R, Ehrhart F, Katsen-Globa A, Zimmermann H, Alves PM, 2009. Cryopreservation of adherent cells: strategies to improve cell viability and function after thawing. Tissue Eng Part C Methods, 15(3):373-86. 
  28. Serra M, Brito C, Costa E, Sousa MF, Alves PM, 2009. Integrating human stem cell expansion and neuronal differentiation in bioreactors. BMC Biotechnol, 9:82. 
  29. Serra M, Brito C, Leite SB, Gorjup E, von Briesen H, Carrondo MJ, Alves PM, 2009. Stirred bioreactors for the expansion of adult pancreatic stem cells. Ann Anat, 191:104-15. 
  30. Malpique R, Katsen-Globa A, Carrondo MJ, Zimmermann H, Alves PM, 2007. Cryopreservation in micro-volumes: impact upon Caco-2 Colon adenocarcinoma cells proliferation and differentiation. Biotechnol Bioeng, 98:155-66. 
  31. Serra M, Leite SB, Brito C, Costa J, Carrondo MJ, Alves PM, 2007. Novel culture strategy for human stem cell proliferation and neuronal differentiation. J Neurosci Res, 85(16):3557-66.