Evaluation of a Miniaturized Biologically Vascularized Scaffold in vitro and in vivo

Abstract

In tissue engineering, the generation and functional maintenance of dense voluminous tissues is mainly restricted due to insufficient nutrient supply. Larger three-dimensional constructs, which exceed the nutrient diffusion limit become necrotic and/or apoptotic in long-term culture if not provided with an appropriate vascularization. Here, we established protocols for the generation of a pre-vascularized biological scaffold with intact arterio-venous capillary loops from rat intestine, which is decellularized under preservation of the feeding and draining vascular tree. Vessel integrity was proven by marker expression, media/blood reflow and endothelial LDL uptake. In vitro maintenance persisted up to 7 weeks in a bioreactor system allowing a stepwise reconstruction of fully vascularized human tissues and successful in vivo implantation for up to 4 weeks, although with time-dependent decrease of cell viability. The vascularization of the construct lead to a 1.5× increase in cellular drug release compared to a conventional static culture in vitro. For the first time, we performed proof-of-concept studies demonstrating that 3D tissues can be maintained within a miniaturized vascularized scaffold in vitro and successfully implanted after re-anastomosis to the intrinsic blood circulation in vivo. We hypothesize that this technology could serve as a powerful platform technology in tissue engineering and regenerative medicine.

Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair

“…MesoT cells were seeded onto the PET membrane and cultured at 37°C and 5% CO 2 using media to generate ECs or EC/SMC mixtures, as noted above, for 28 days under static conditions in a standard well-plate. Primary dermal microvascular endothelium, a gift from Heike Walles (Kress et al, 2018), cultured in VascuLife® VEGF-Mv Endothelial Complete Kit (Lifeline Cell Technology, LL-0005) were used as a control. Barrier integrity of cells was examined by trans -endothelial electrical resistance (TEER) measurements and a FITC-dextran (40 kDa, Sigma-Aldrich, FD40S) permeability assay.…”

“…Jejunal scaffolds were isolated as described previously (Kress etal., 2018). Briefly, 6-8 week old anesthetized Lewis rats were subject to a median laparotomy to isolate a jejenal segment containing its arterial and venous pedicle.…”

“…Following a one-hour static incubation, allowing for cellular adherence, the injection and static incubation were repeated. To promote functional maturation of the cellular layer on the vascular bed, the scaffold was connected to a bioreactor system to mimic physiological blood flow conditions by perfusion culture (Kress et al, 2018). Cell injection and static adherence were repeated once per arterial and venous cannula per day with subsequent perfusion culture overnight for three consecutive days.…”

“…FITC-dextran retention and vascular integrity was detected using real-time fluorescence as previously described (Kress et al, 2018). Functional endothelium lining the vascular tree was confirmed via acetylated LDL uptake (Invitrogen, L3484); nuclei were counterstained with NucBlue Live ReadyProbes (Invitrogen, R37605).…”

3D organ models—Revolution in pharmacological research?

“…brain) tissues, specialized techniques such as high-pressure water jet cutting need to be employed. Interestingly, such tissues may be integrated more easily into perfusion chambers or microfluidic systems for preclinical studies that may increase their possible cultivation periods [46]. Tissue sections reflect the spatiotemporal behavior of functional units within human organs without the inclusion of systemic body responses such as immune cell recruitment or hormonal effects.…”