Bioprinting of high cell‐density constructs leads to controlled lumen formation with self‐assembly of endothelial cells

Abstract

Active nutrient supply and waste product removal are key requirements for the fabrication of long‐term viable and functional tissue constructs of considerable size. This work aims to contribute to the fabrication of artificial perfusable networks with a bioprinting process, based on drop‐on‐demand (DoD) printing of primary endothelial cell (EC) suspension bioink (25 × 106 ± 3 × 106 cells/ml). The process results in prescribed lumen between two hydrogel layers, allowing its integration in common layering based bioprinting processes. Low volume bioink droplets (appr. 10 nl) as building blocks were deposited between two fibrin or Collagen I layers to realize shapeable, cell‐rich aggregates. Unattainable with manual positioning, DoD printing allowed precise fabrication of various designs, such as spheroidal‐, line‐shaped, and Y‐branch cellular structures, with a mean lateral extension of 285 ± 81 μm. For basic characterization, the cell suspension building blocks were systematically compared with preformed spheroids of the same cell type, passage, and number. Post printing investigations of initially loose cell arrangements showed self‐assembly and formation of central lumen with a mean cross‐sectional area of Ølumen = 6,400 μm2 at Day 3, lined by a single layer of CD31 positive ECs, as evaluated by confocal microscopy. Originating from this main lumen smaller, undirected side branches (Øbranches = 740 μm2) were formed by sprouting cells, inducing a first step towards a simplistic hierarchically organized network. These lumen could prospectively help for tissue construct perfusion in vitro or, potentially, as niche for angiogenesis of host vascularization in implants.

In vivo evaluation of bioprinted prevascularized bone tissue

“…This approach focuses on self-assembly of HUVECs to generate blood vessels upon implantation. We have previously shown in vitro that this approach leads to the formation of lumenized vessel-like structures (Trondle et al, 2019). Based on these in vitro experiments, we have now tested bioprinted HUVECs for their ability to form functional blood vessels in vivo in a subcutaneous implantation model.…”

“…We have previously identified specific hydrogels that support various and crucial cell parameters of ASCs and HUVECs such as viability, differentiation, bone matrix formation and endothelial sprouting in vitro (Benning et al, 2017, 2018; Gutzweiler et al, 2017; Trondle et al, 2019; Wehrle et al, 2019). This led to the identification of a complex hydrogel consisting of fibrinogen, glycerol, hyaluronic acid, and gelatin that was described already by Kang et al (2016).…”

“…In recent studies, we have identified suitable hydrogels for extrusion‐based bioprinting of human adipose‐derived mesenchymal stem cells (ASCs) and DoD‐based bioprinting of HUVECs (Trondle et al, 2019; Wehrle et al, 2019). In this context, we have shown in vitro that a multicomponent hydrogel with a thermal and enzymatic crosslinking mechanism due to gelatin and fibrin showed excellent biocompatibility toward ASCs.…”

“…Similarly, we could show that HUVECs bioprinted via DoD using fibrin hydrogel and a high cell concentration showed a high cell viability after printing of more than 80%. Moreover, HUVECs displayed a time‐dependent self‐assembly of lumenized vascular structures in vitro (Trondle et al, 2019).…”

Effects of Processing Parameters of 3D Bioprinting on the Cellular Activity of Bioinks

“…High density promotes self-assembly of endothelial cells and is favorable for the generation of controlled lumens that helps in the vascularization of implants. [117] Hence, it can be inferred that the selection of cell density varies with cell type. Based on the literature, Cidonio et al mentioned that although Figure 10.…”