Doctoral thesis

Injectable biomaterial for in situ platelet production: in vitro support advancements and in vivo vascularization study

ContributorsBrefie-Guth, Joe
Imprimatur date2023
Defense date2023

Platelet transfusion is a life-saving procedure to reduce the bleeding risk of patients or in patients with platelet function-related disorders. The majority of platelet transfusion is prescribed to hemato-oncologic patients, especially acute leukaemias and myelodysplastic syndrome patients, who have compromised haematopoiesis.

Platelets are cytoplasmic fragments of mature haematopoietic cells called megakaryocytes. They are a key player in the process of blood clotting and are produced physiologically in the bone marrow from megakaryocyte membrane projections that release the platelets into the bloodstream via specialized sinusoidal vessels.

This PhD project aimed at determining the conditions to provide a supportive niche for megakaryocytes to survive as a highly vascularized subcutaneous injectable implant that could be further developed for in vivo platelet production in these fragile patients, by bridging the expertise in material bioengineering and bone marrow microenvironment of, the Prof. Braschler lab at the University of Geneva and the Prof. Naveiras lab at the University of Lausanne, respectively.

To develop this innovative combined therapy, we used a functionalized carboxymethyl cellulose-based scaffold compatible with haematopoietic progenitor cells adhesion and vascularization during subcutaneous transplantation. However, this approach had not been tested for megakaryopoiesis.

This thesis focused on three objectives: (1) the functional support of megakaryopoiesis and platelet production in vitro, (2) in vivo bare scaffold implantation, and finally (3) the vascularization of a megakaryocyte implant.

To optimize functional support, megakaryocytes were differentiated on different protein supports prior to platelet production. We showed that 3D culture had no impact on platelet production compared to 2D culture and that protein coatings of the extracellular matrix favoured the production of functional platelets. In addition, we have developed a perfusion system that mimics the physiological shear conditions contained in the sinusoids to further characterise the protein coating.

By studying and optimizing the biomaterial injectability and its biointegration in vivo, we were able to characterize the properties of the biomaterial for applications in tissue engineering. The analysis of the transcriptome by single-cell RNA sequencing methods of the cells colonizing the bare scaffold after implantation made it possible to validate its biocompatibility but also to observe its low angiogenic potential.

Finally, vascularization is intended to promote cell egression. To enhance vascularization, we studied the ability of various populations of haematopoietic cells or proteins to induce and regulate vessels in the implant. We loaded the niche with different cell types and transplanted subcutaneously into SCID NOD mice. We then studied the vascular

Citation (ISO format)
BREFIE-GUTH, Joe. Injectable biomaterial for in situ platelet production: <i>in vitro</i> support advancements and <i>in vivo</i> vascularization study. 2023. doi: 10.13097/archive-ouverte/unige:177573
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Creation05/31/2024 3:24:26 PM
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