Advanced Cell Models for pre-clinical research

As befits a technological area, the Advanced Cell Models Laboratory is integrated in the Animal Cell Technology Unit, where a number of knowledge competences are balanced.

Our research is mostly translational, focused on the study of cellular microenvironment in disease progression and therapeutic response. The major challenge in studying these mechanisms is the lack of human cell models in which the different contributing cell types are represented and the dynamics of the cellular and extracellular spaces recapitulated along time. To overcome these challenges we develop innovative disease cell models by applying advanced cell culture approaches (3D culture, co-culture, cell immobilization) and systems (bioreactors) to human stem cells and other patient-derived cells. By challenging these models with therapeutic agents and integrating biochemical, cell imaging and omics approaches we are moving towards the definition of the role of the microenvironment to therapeutic response. We aim at the identification of novel molecular players potentially involved in therapeutic response, towards novel biomarkers or therapeutic targets.

Cancer Cell Models: we have been developing cell models in which tumor-stromal crosstalk and disease progression events are recapitulated, with epithelial tumor cells surrounded by fibroblasts and immune cells, and deposition of secreted factors and extracellular matrix components. Recently, within the scope of iNOVA4Health, we are developing ex-vivo models by implementing long-term cultures of tumor explants, focusing on colorectal, ovarian and breast cancer.

Neural Cell Models: we are applying patient-derived 3D neural cell models to explore the influence of extracellular space defects in mechanisms involved in disease progression, leading to neuronal and synaptic functional impairment and neurodegeneration. We employ induced pluripotent stem cell (iPSC) technology and of a novel culture strategy for 3D neural differentiation of human neural stem cells (hNSC) into neurons, astrocytes and oligodendrocytes, recently develop by us.


Selected Publications


  1. 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. 
  2. Santo VE, Rebelo SP, Estrada MF, Alves PM, Boghaert E, Brito C. 2017. Drug screening in 3D in vitro tumor models: overcoming current pitfalls of efficacy read-outs. Biotechnology Journal.  
  3. Stock K, Estrada MF, Vidic S, Gjerde K, Rudisch A, Santo VE, Barbier M, Blom S, Arundkar SC, Selvam I, Osswald A, Stein Y, Gruenewald S, Brito C, van Weerden W, Rotter V, Boghaert E, Oren M, Sommergruber W, Chong Y, de Hoogt R, Graeser R. 2016. Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery. Scientific Reports. 6, 28951.  
  4. Barbier M, Jaensch S, Cornelissen F, Vidic S, Gjerde K, de Hoogt R, Graeser R, Gustin E, Chong YT; Hickman J, Burbridge M, Verschuren E, Kallioniemi O, Klefström J, Brisken C, Rotter V, Oren M, Brito C, Schalken J, Jenster G, van Weerden W, Vilo J, Schueler J, Monni O, Barry ST, Grünewald S, Kallio P, Mueller H, Nopora A, Sommergruber W, Anderson E, van der Kuip H, Smalley M,  Boghaert E. 2016. Ellipsoid Segmentation Model for Analyzing Light-Attenuated 3D Confocal Image Stacks of Fluorescent Multi-Cellular Spheroids.  PLoS One .  11, e0156942. 
  5. Terrasso AP, Silva AC, Filipe A, Pedroso P, Ferreira AL, Alves PM, Brito C. 2016. Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.  J Pharmacol Toxicol Methods. 83:72-79. 
  6. Simão D, Terrasso AP, Teixeira AP, Brito C, Sonnewald U, Alves PM. 2016. Functional metabolic interactions of human neuron-astrocyte 3D in vitro networks.  Scientific Reports. 6, 33285. 
  7. Santo VE, Estrada MF, Rebelo SP, Abreu S, Silva I, Pinto C, Veloso SC, Serra AT, Boghaert E, Alves PM, Brito, C. 2016. Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models. J Biotechnol. 221 118-29. 
  8. Estrada MF, Rebelo SP, Davies EJ, Pinto MT, Pereira H, Santo VE, Matthew J. Smalley, Barry ST, Gualda EJ, Alves PM, Anderson E, Brito C. 2016. Modelling the tumor microenvironment in long-term microencapsulated 3D cocultures recapitulates phenotypic features of disease progression. Biomaterials (in press). 
  9. Simao D, Pinto C, Fernandes P, Piersanti S, Saggio I, Collinson LM, Schiavo G, Kremer EJ, Alves PM & Brito C. 2016. Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model. Gene Therapy, 23(1):86-94 
  10. Materne EM, Ramme AP, Terrasso AP, Serra M, Alves PM, Brito C, Sakharov DA, Tonevitsky AG, Lauster R, Marx U. 2015. A multi-organ chip co-culture of neurospheres and liver equivalents for long-term substance testing. J Biotechnol. Feb 9. pii: S0168-1656(15)00045-0. 
  11. 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]
  12. Terrasso AP, Pinto C, Serra M, Filipe A, Almeida S, Ferreira AL, Pedroso P, Brito C, Alves PM. 2015. Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints. J Biotechnol. 2015 Jan 5. pii: S0168-1656(14)01047-5.
  13. Rebelo SP, Costa R, Estrada M, Shevchenko V, Brito C, Alves PM. 2014. HepaRG microencapsulated spheroids in DMSO‑free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism. Archives of Toxicology. Published on line 09 August 2014. 
  14. 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. 2014. Modelling human neural functionality in vitro: 3D culture for dopaminergic differentiation. Tissue Engineering – Part A, Epub ahead of print, Sep 25. 
  15. Hickman J, Graeser R, De Hoogt R, Vidic S, Brito C, Gutekunst M, Van der Kuip H. 2014. Capturing tumor complexity in vitro/ex vivo: three dimensional models of cancer for pharmacology and cancer cell biology. Biotechnology Journal, 9: 1115-28. 
  16. Sarmento L, Póvoa V, Nascimento R, Real G, Antunes I, Martins L, Moita M, Alves PM, Abecasis M, Moita L, Parkhouse R, Meijerink J and Barata JT. 2014. CHK1 overexpression in T-cell acute lymphoblastic leukemia is essential for proliferation and survival by preventing excessive replication stress. Oncogene. 2014 Aug 18;0. 
  17. Rebelo SP, Costa R, Estrada M, Shevchenko V, Brito C, Alves PM. 2014. HepaRG microencapsulated spheroids in DMSO‑free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism. Archives of Toxicology. Published on line 09 August 2014. 
  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 online 6 August 2014.  
  20. Tostões R, Leite SB, Serra M, Jensen J, Björquist P, Carrondo MJ, Brito C, Alves PM, 2012. Human liver cell spheroids in extended perfusion bioreactor culture for repeated dose drug testing. Hepatology, 55(4): 1227-36. 
  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. Almeida AS, Queiroga CS, Sousa MF, Alves PM, Vieira HL, 2012. Carbon monoxide modulates apoptosis by reinforcing oxidative metabolism in astrocytes: role of BCL-2. J Biol Chem, 287 (14): 10761-70. 
  23. Brito C, Simão D, Costa I, Malpique R, Pereira CI, Fernandes P, Serra M, Schwarz SC, Schwarz J, Kremer EJ, Alves PM, 2012. Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells. Methods, 56(3):452-60. 
  24. Leite SB, Wilk-Zasadna I, Zaldivar Comenges JM, Airola E, Reis-Fernandes MA, Mennecozzi M, Guguen-Guillouzo C, Chesne C, Gouillou C, Alves PM, Coecke S, 2012. 3D HepaRG model as an attractive tool for toxicity testing. Toxicol Sci 130 (1): 106-116. 
  25. 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.
  26. Vieira HL, Alves PM, Vercelli A, 2011. Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species. Prog Neurobiol, 93:444-55. 
  27. 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. 
  28. Tostões RM, Leite SB, Miranda JP, Sousa MF, Wang DIC, Carrondo MJ, Alves PM, 2011. Perfusion of 3D encapsulated hepatocytes-a synergistic effect enhancing long-term functionality in bioreactors. Biotechnol Bioeng, 108: 41-49. 
  29. Santos SS, Sonnewald U, Carrondo MJ, Alves PM, 2011. The role of glia in neuronal recovery following anoxia: in vitro evidence of neuronal adaptation. Neurochem Int, 58:665-75. 
  30. Leite SB, Teixeira AP, Miranda JP, Tostoes RM, Clemente JJ, Sousa MF, Carrondo MJ, Alves PM, 2011. Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: improved in vitro models for Toxicological applications. Toxicol in Vitro, 25(4):825-32. 
  31. Queiroga CS, Almeida AS, Alves PM, Brenner C, Vieira HL, 2011. Carbon monoxide prevents hepatic mitochondrial membrane permeabilization. BMC Cell Biol 12:10. 
  32. Amaral AI , Teixeira AP, Sonnewald U, Alves PM, 2011. Estimation of intracellular fluxes in cerebellar neurons after hypoglycemia: importance of the pyruvate recycling pathway and glutamine oxidation. J Neurosci Res, 89(5)-700:10. 
  33. Amaral IA, Teixeira AP, Håkonsen BI, Sonnewald U, Alves PM, 2011. A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and 13C-labelled glucose. Front Neuroenerg, 3:5. 
  34. Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Andersen KE, Angers-Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Mazzatorta P, Maxwell G, Munn S, Leite SB, Loizou G, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tähti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM, 2011. Alternative (non-animal) methods for cosmetics testing: current status and future prospects. Arch Toxicol, 85(5):367-485. 
  35. Amaral AI, Teixeira AP, Martens S, Sousa MF, Bernal V, Alves PM, 2010. Metabolic alterations induced by ischemia in primary cultures of astrocytes: merging 13C NMR spectroscopy and metabolic flux analysis. J Neurochem, 113(3):735-48. 
  36. Miranda JP, Rodrigues A, Tostões RM, Leite SB, Zimmerman H, Carrondo MJ, Alves PM, 2010. Extending hepatocyte functionality for drug-testing applications using high-viscosity alginate-encapsulated three-dimensional cultures in bioreactors. Tissue Eng Part C Methods, 16(6):1223-32. 
  37. 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. 
  38. 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. 
  39. Fortalezas S, Tavares L, Pimpão R, Tyagi M, Pontes V, Alves PM, McDougall G, Stewart D, Ferreira RB, Santos CN, 2010. Antioxidant properties and neuroprotective capacity of strawberry tree fruit (Arbutus unedo). Nutrients, 2(2):214-29. 
  40. Miranda JP, Leite SB, Muller-Vieira U, Rodrigues A, Carrondo MJ, Alves PM, 2009. Toward extended functional hepatocyte in vitro culture. Tissue Eng Part C Methods, 15:1-11. 
  41. 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. 
  42. 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. 
  43. 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. 
  44. Güll I, Alves PM, Gabor F, Wirth M, 2009. Viability of human adenocarcinoma cell line Caco-2: influence of cryoprotectant, freezing rate, and storage temperature. Sci Pharm, 77:133-41. 
  45. Vieira HL, Queiroga CS, Alves PM, 2008. Preconditioning induced by Carbon Monoxide provides neuronal protection against apoptosis. J Neurochem, 107:375-84. 
  46. Teixeira AP, Santos SS, Carinhas N, Oliveira R, Alves PM, 2008. Combining metabolic flux analysis tools and 13C-NMR to estimate intracellular fluxes of cultured astrocytes. Neurochem Int, 52:478-86. 
  47. Petrovski Z, Norton de Matos MR, Braga SS, Pereira CC, Matos ML, Gonçalves IS, Pillinger M, Alves PM, Romão CC, 2008. Synthesis, characterization and antitumor activity of 1,2-disubstituted ferrocenes and cyclodextrin inclusion complexes. J Organomet Chem, 693:675-84. 
  48. 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. 
  49. Santos SS, Leite SB, Sonnewald U, Carrondo MJ, Alves PM, 2007. Stirrer vessel cultures of rat brain cells aggregates: characterization of major metabolic pathways and cell population dynamics. J Neurosci Res, 85(15):3386-97. 
  50. 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. 
  51. Santos SS, Gibson GE, Cooper AJ, Denton TT, Thompson CM, Bunik VI, Alves PM, Sonnewald U, 2006. Inhibitors of the a-ketoglutarate dehydrogenase complex alter [1-13C]Glucose and [U-13C]Glutamate metabolism in cerebellar granule neurons. J Neurosci Res, 83:450-8. 
  52. Fonseca LL, Monteiro MA, Alves PM, Carrondo MJ, Santos H, 2005. Cultures of rat astrocytes challenged with a steady supply of glutamate: a new model to study flux distribution in the glutamate-glutamine cycle. Glia, 51:286-96. 
  53. Santos SS, Fonseca LL, Monteiro MA, Carrondo MJ, Alves PM, 2005. Culturing primary brain astrocytes under fully controlled environment in a novel bioreactor. J Neurosci Res, 79:26-32. 
  54. Alves PM, Fonseca LL, Peixoto CC, Almeida AC, Carrondo MJ, Santos H, 2000. NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia. NMR Biomed, 13:438-48. 
  55. Alves PM, Nunes R, Zhang C, Maycock CD, Sonnewald U, Carrondo MJ, Santos H, 2000. Metabolism of 3-[13C]malate in primary cultures of mouse astrocytes. Dev Neurosci, 22:456-62. 
  56. Alves PM, JL Moreira, JM Rodrigues, JG Aunins, MJ Carrondo, 1996. Two-dimensional versus three-dimensional culture systems: effects on growth and productivity of BHK cells. Biotechnol Bioeng, 52:429-32. 
  57. Alves PM, Moreira JL, Rodrigues JM, Aunins JG, Carrondo MJ, 1996. Immobilization of primary astrocytes and neurons for on-line monitoring of biochemical processes by NMR. Dev Neurosci, 18:478-83. 
  58. Alves PM, MC Mckenna, U Sonnewald, 1995. Lactate metabolism in mouse brain astrocytes studied by [13C]NMR spectroscopy. NeuroReport, 6:2201-04. 
  59. Moreira JL, Cruz PE, Santana P, Aunins JG, Carrondo MJ, 1995. Formation and disruption of animal cell aggregates in stirred vessels: mechanisms and kinetic studies. Chem Eng Sci, 50:2747-64. 
  60. Moreira JL, Alves PM, Aunins JG, Carrondo MJ, 1994. Changes in animal cell natural aggregates in suspended batch cultures. Appl Microbiol Biotechnol, 41: 203-9. 
  61. Moreira JL, Alves PM, Rodrigues JM, Cruz PE, Aunins JG, Carrondo MJ, 1994. Studies of baby hamster kidney natural cell aggregation in suspended batch cultures. Ann N Y Acad Sci, 745:122-33. 
  62. Moreira JL, Feliciano AS, Santana PC, Cruz PE, Aunins JG, Carrondo MJ, 1994. Repeated-batch cultures of baby hamster kidney cell aggregates in stirred vessels. Cytotechnology, 15:337-49.