Sistemas 3d para el cultivo de células inmunes y sus aplicaciones en inmunoterapia
- Autores: Eduardo Perez del Rio
- Directores de la Tesis: Imma Ratera Bastardas (dir. tes.), Judit Guasch Camell (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Miguel Ángel Mateos Timoneda (presid.), Carlos Mas Moruno (secret.), Beatriz Morancho Armisen (voc.)
- Programa de doctorado: Programa de Doctorado en Bioquímica, Biología Molecular y Biomedicina por la Universidad Autónoma de Barcelona
- Materias:
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- Resumen
Recent achievements in the field of immunotherapy, such as the development of engineered T cells used in adoptive cell therapy, are introducing more efficient strategies to combat cancer. Nevertheless, these T cells are challenging to manufacture, manipulate, and control. Specifically, there are limitations in producing the large amounts of T cells needed for these therapies in a short period of time and in an economically viable manner. In this thesis, we have studied different 3D systems with the objective of achieving higher proliferation rates and tune the resulting phenotypes, resembling the natural environment of the secondary lymphoid organs.
Matrigel and a 3D polystyrene scaffold were studied as two of the most suitable commercially available options, showing an increase in cell proliferation compared to standard suspension systems. This research proved not only the beneficial effect of the addition of a 3D physical support, but also the importance of the chemical input to stimulate cell expansion. However, these systems were not designed for secondary lymphoid organ mimicking and thus, they significantly differ in terms of ECM composition. Consequently, a new platform was developed for this application to improve the obtained results.
Hydrogels composed of poly(ethylene) glycol (PEG) covalently combined with low molecular weight heparin (PEG-Hep hydrogels) were synthesized and completely characterized for this purpose. PEG confers the 3D structure to the hydrogel, and can be manipulated to change its mechanical properties. Heparin, which is a negatively charged molecule, is used as an anchor for positively charged proteins that can affect cell behavior. This system was used for immune cell expansion under different conditions, specifically, unloaded hydrogels were employed as well as hydrogels loaded with cytokines related to immune cell proliferation like CCL21 and CCL19. All the conditions resulted in an increase of CD4+ T cell proliferation but the highest proliferation indexes were achieved with hydrogels loaded with CCL21 together with the addition of CCL19 in suspension, which are the conditions that best mimic the extracellular matrix of lymph nodes. Besides, the resulting phenotypes of CD4+ T cells cultured in PEG Hep hydrogels showed an increase of the TEM percentage in comparison with cells expanded in suspension. The same systems were used for peripheral blood mononuclear cells (PBMCs). 3D polystyrene improved the proliferation parameters in comparison with suspension systems. However, the killing capacity of the resulting cells did not change in comparison with cells cultured in suspension due to the lack of chemical input in this system. The beneficial results obtained with PEG-Hep hydrogels for the culture of CD4+ T cells could not be reproduced for this population. Nevertheless, PEG-Hep hydrogels did show an effect in the resulting phenotypes achieved, increasing the CD45RO+/CD62L- percentage (TEM population for T cells). Finally, PEG-Hep hydrogels were studied as bioink for 3D printing, optimizing the protocol of hydrogel formation for this application which required a scaling up process. The resulting printed scaffolds were applied to immune cell culture. It was observed increases in the proliferation parameters of CD4+ T cells and changes in their resulting phenotypes in comparison with non-printed hydrogels. In this case an increase in the TCM phenotype was observed, which is related to good clinical outcomes. For PBMCs no proliferation enhancement was observed and the resulting phenotypes were comparable with the ones obtained without printing. However, a further optimization of the use of PEG-Hep hydrogels as bioink for 3D printing is still necessary.
