Speaker
Description
While three dimensional-bioprinting has already gained global attention as a rapidly advancing field, the creation of properly vascularized tissues remains challenging. Extrusion-based bioprinting is specifically interesting for the fabrication of large tissues, but has a limited resolution. Therefore, with extrusion-based bioprinting, microvasculature formation through self-assembly is currently being explored. In this research project, the potential of self-amplifying mRNA (sa-mRNA) engineered cells in promoting microvascular self-assembly was explored. Sa-mRNA is especially interesting due to its prolonged activity compared to other synthetic mRNAs. A DNA template for VEGF-producing self amplifying mRNA (VEGF-sa-mRNA) was generated by inserting the VEGF165A sequence into a Venezuelan Equine Encephalitis Virus (VEEV)-derived saRNA template plasmid using NEBuilder® HiFi DNA Assembly (NEB). Following linearization, in vitro transcription, and capping of the VEGF-sa-mRNA, cells were transfected with Lipofectamine® MessengerMAX reagent (Invitrogen). First, the VEGF production by the transfected cells was verified together with its functionality. This was done by incubating a co-culture of human adipose stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) in endothelial medium supplemented with the conditioned medium of baby hamster kidney cells (BHKs), transfected with the VEGF-sa-mRNA. A 7 day incubation in the supplemented medium resulted in significantly higher network length compared to negative control. In a follow-up experiment, a novel scaffold consisting of a recently reported porous bioink was bioprinted1. Within this scaffold, ASCs/HUVECs spheroids were combined with VEGF-sa-mRNA transfected BHKs. After 7 days in culture, vascular spheroids in this construct showed a significant increase in capillary sprouting compared to a negative control lacking the transfected cells. In the context of clinical translatability of the VEGF-sa-mRNA system, transfection of human ASCs was explored as well. More specifically, the ability of an additional cellulose purification step in removing double stranded RNA strands was investigated to reduce the cellular innate immune response and increase sa-mRNA activity. Additionally, the effect of transfection duration on the viability and protein expression of ASCs was analysed. Finally, the VEGF production by these transfected ASCs was monitored across different time periods using ELISA. Cellulose purified sa-mRNA resulted in a significant increase in protein production compared to non-cellulose purified sa-mRNA. A Transfection duration limited to 30 mins was superior in both viability and protein expression. A more detailed analysis using ELISA demonstrated that VEGF production starts as early as 2 hours post-transfection, reaching its peak at 18 hours (59,49 ng/mL, produced by 40,000 ASCs). In summary, this research project took the first steps towards evaluating the potential of sa-mRNA engineered ASCs in stimulating the self-assembly of microvasculature. Future prospects include evaluating the functionality of VEGF produced by transfected ASCs within a bioprinted scaffold. The established VEGF-sa-mRNA system could play a crucial role in applications within regenerative medicine and patient-specific 3D-bioprinted organ models.
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