Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Cardiovascular Engineering

Cardiovascular Engineering - Poster Session A

Poster H8 - Biomanufacturing of stromal cell-reinforced patterned vessel channels for regenerative medicine

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  In the US, someone experiences a heart attack once every 40 seconds. Given complications such as scarring and impaired myocardial contractility resulting from infarct-related cell death, research aims to replace lost cardiomyocytes through the delivery of human induced pluripotent stem cell-derived cardiomyocytes. One method of delivering cells includes encapsulation in a collagen scaffold to produce engineered human myocardium (EHM). Implanted EHM relies on vascularization by the host circulatory system for survival and engraftment. Research has shown that incorporating endothelial cells (ECs) in pre-patterned arteriole-scale vessel channels can facilitate rapid inosculation and integration with the host vasculature. Similarly, in vitro perfusion of the patterned vessels prior to implant further improves both cardiomyocyte and endothelial cell engraftment and integration. However, several obstacles persist. Manually fabricating and coating these EC-lined channels can be a variable process. Additionally, barrier function and stability of these vessels can be limited by their lack of the vascular stromal support cells (vSCs) seen in vivo. This work aims to improve the consistency and function of patterned vessels for engineered tissues. By introducing a 3D-printed frame to guide fiber placement and improving the compliance of the gelatin-alginate-collagen (GAC) gel used to create endothelialized channels, the procedure is made more consistent. Additionally, vSCs and enhanced shear-conditioning will be used to improve vessel wall structure and barrier function.

Materials and

Methods:  EHM with endothelialized channels are developed by threading sacrificial (to be later removed) gelatin-alginate fibers through a polymer tissue mold and then coating the fibers with a GAC gel containing vascular ECs derived from induced pluripotent stem cells with or without supplemental vSCs. Once set, the coated fibers are cast in a collagen scaffold. The gelatin-alginate fiber is uncrosslinked when proper hydrogel integrity is achieved, producing a patent endothelialized channel. The channel inlets are then connected to a peristaltic pump to begin perfusion culture and shear conditioning of the endothelial barrier (Figure 1A). The tissues are perfused for five days, and media flow rates are chosen based on a computational fluid dynamics model assessing corresponding wall shear stress and nutrient diffusion. When adjusting the GAC properties, collagen concentrations were changed from 4 mg/mL to both 2 mg/mL and 8 mg/mL. Endothelialized channels containing either ECs alone or ECS with vSCs were perfused under the same conditions. Barrier functions from the two groups were compared by adding dye to the perfusion media on day 5 of perfusion and observing diffusion. To better assess the barrier function, post-perfusion tissues are attached to a syringe pump, where media containing Evans Blue dye can be pushed through the channel at a controlled volume and speed. Imaging of the dye to assess penetration into the tissue allows for quantification of barrier function, and immunohistochemical (IHC) staining of ECs, vSCs, tight junctions, and deposited basement membrane proteins enable assessment of vessel wall morphology.

Results, Conclusions, and Discussions: It was found that decreasing the concentration of collagen within the GAC gel from 4 mg/mL to 2 mg/mL successfully reduced abnormal channel formation while maintaining the gelation properties of the gel. While data collection is still ongoing, perfusion trials show that endothelialized channels with vSCs exhibit improved barrier function and vessel wall morphology when compared to channels containing only ECs (Figure 1 D, E). Using our barrier function quantification model and the results from the pilot coating experiments, we expect to see reduced leakiness associated with vSC-incorporated endothelialized channels. Based on the computational fluid dynamics model, we also expect endothelial integrity to improve upon increasing the flow rates used for shear conditioning. These results will inform our ability to improve EHM fabrication with endothelialized channels to further support its survival upon implant and integration with a post-infarct heart.

Acknowledgements (Optional):