3D biohybrid hydrogels as artificial extracellular matrix for CAR T cell and organoid culture

Abstract

This thesis describes the development of novel biohybrid hydrogels consisting of poly(ethylene glycol) – heparin as artificial extracellular matrices for applications in immunotherapy and cancer modelling. In particular, we focused on improving the ex vivo manufacture of CAR T cell products for cancer immunotherapy, and the establishment of both hematological and solid cancer organoid (tumoroid) models. With these objectives, efforts were directed to optimizing not only the mechanical and structural properties of the biohybrid hydrogels, but also the biochemical ones by the addition of relevant biomolecules through different chemical strategies. Moreover, we also assessed the introduction of a dynamic control into the hydrogels by a supramolecular reversible crosslinking. _x000D_ More specifically, chapter 2 shows the preparation and characterization of the biohybrid hydrogels developed and used in this thesis. The synthesis of maleimide functionalized heparin (Hep) and its crosslinking with 4-arm thiolate polyethylene glycol (PEG) to form the standard hydrogels is shown, together with the different modifications performed. A multi-technique characterization approach was followed for all described hydrogels._x000D_ In chapter 3, the physicochemical characteristics of novel PEG-Hep hydrogels with lymph-node mimicking features are studied and optimized for the ex vivo manufacturing of CAR T cell products. This chapter includes one of the compiled published articles of this thesis. _x000D_ In chapter 4, the importance of biomolecules, namely cytokines and adhesion molecules, in the modulation of immune functions through primary human T cell behavior is discussed. In particular, PEG-Hep hydrogels are functionalized by electrostatic interactions with cytokines (CCL21, CCL19) and adhesion molecules (ICAM-1) to mimic the lymph nodes, and their impact on T cell proliferation is assessed. Surprisingly, phosphate ions present in biological buffers and cell media were seen to play a key role in the interaction between the cytokine CCL21 and heparin._x000D_ In chapter 5, hydrogels comprising supramolecular crosslinks were assessed to introduce dynamic properties into the system and be able to obtain a reversible hydrogel. The strategy chosen to achieve this goal was a host-guest reaction consisting of phenylalanine derivatives (Phe) linked to the hydrogel backbone as guest molecules and cucurbit[8]uril (CB8) as a supramolecular host._x000D_ In chapter 6, the advantages of biohybrid hydrogels, comprising both synthetic and natural origin polymers, as artificial extracellular matrices for organoid and tumoroid culture are discussed. This chapter includes the second of the compiled published articles of this thesis. _x000D_ Finally, in chapter 7, biohybrid hydrogels are assessed for the culture of both hematological and solid cancer organoid models. Specifically, colorectal and follicular lymphoma cancer models consisting of patient-derived cancer organoids are evaluated using PEG-Hep hydrogels._x000D_ The last chapter is dedicated to the conclusions and future perspectives for this thesis. Additionally, my scientific trajectory is included to explain complementary activities performed during this thesis such as conference attendance or master student supervision._x000D_ In summary, the work of this PhD thesis contributes to the development of advanced biomaterials consisting of biohybrid hydrogels for oncological applications. In particular, tissue mimicking hydrogels were explored for two complementary applications in promising and growing fields such as immunotherapy and organoid cancer models.

Date of Award	27 Nov 2025
Original language	English
Awarding Institution	Universitat Autònoma de Barcelona (UAB)
Supervisor	Judit Guasch Camell (Director)