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Köpf, et al.
printing resolution and the mechanisms governing how 2. Materials and methods
vascular structures are generated as well as their size [8,9] .
However, in most bioprinting procedures the cells are 2.1. Cell isolation
subjected to shear stress, which in turn diminishes Human mesenchymal stromal cells (hMSCs) were
cell viability after printing and potentially also affects isolated from the femoral heads of patients receiving hip
post-printing cell functionality . However, because joint arthroplasty after informed consent approved by the
[10]
the amplitude and time course of shear stress can vary Ethics Committee of the Faculty of Medicine, RWTH
among these techniques, it is essential to fully understand Aachen University (EK 300/13). A cell suspension
potential side effects of the printing process on the cells. was prepared by flushing the spongiosa several times
Investigating the impact of printing-induced shear stress with hMSC growth medium (Mesenpan, PAN Biotech,
on cells should involve a rheological characterization of Aidenbach, Germany) containing 2% v/v fetal calf serum
printing suspensions, a simulation and quantification of the (PAN Biotech, Aidenbach, Germany), 1% v/v penicillin/
stress condition for the cells during the printing process, streptomycin (Gibco, Invitrogen, Carlsbad, USA), and
and extensive, conclusive, and thoroughly planned cell 1% v/v growth supplement (PAN Biotech, Aidenbach,
culture studies specifically designed with respect to cell Germany). After centrifugation (1,200 rpm for 5 min;
and tissue type. In this study considering the geometry of CT6EL, Hitachi Koki, Tokyo, Japan) and removal of
a commonly used mechanical microvalve, we performed the supernatant, the cells were plated inside two T75
a finite element analysis (FEA) simulation to analyze the culture flasks (CELLSTAR TC, sterile, Greiner Bio-One,
mechanical load in terms of shear stress and hydrostatic Frickenhausen, Germany). The flasks were incubated at
pressure during drop ejection in DoD bioprinting. As an 37°C und 5% v/v CO . Non-adherent cells were removed
2
input material characteristic, the measured viscosity of during the first media change after 24 h. Starting that day,
alginate solution at a concentration of 1.5% (w/v) is used the media were changed twice a week.
and a power-law function is applied to model its non- HUVECs were isolated from umbilical cords
Newtonian shear-thinning behavior. and provided by the Department of Gynecology and
Blaeser et al. previously reported a theoretical Perinatal Medicine (RWTH Aachen University Hospital)
model based on simplified assumptions and geometry as approved by the local ethics committee (EK 218/14).
to calculate the induced shear stress during DoD HUVECs were isolated following established protocols [6,7] .
bioprinting . However, our FEA-based simulation Briefly, the umbilical cords were rinsed in phosphate-
[10]
model in the present paper considers the actual geometry buffered saline (PBS) for 5 min. To remove coagulated
of the printing valve (including moving and stationary blood, the veins were flushed with PBS and then filled
pistons and conical shape of nozzle inlet with actual with collagenase solution (Collagenase Type I, 400 U/
curves) and therefore provides more reliable results as ml dissolved in Hank’s Balanced Salt Solution + CaCl , +
2
well as a more complete picture of the shear stress which MgCl , both Gibco by Life Technologies, Carlsbad, USA)
2
is dependent on the applied upstream pressure during and closed with a clip at both ends. The umbilical cord
the bioprinting process. Moreover, our developed model was then placed on a petri dish, covered, and incubated for
enabled us to calculate flow parameters in each single 30 min (37°C and 5 % v/v CO ). The clips were removed
2
geometrical point; this is not possible using analytical and fresh PBS was used to flush the vein. The cell
integral models. In addition to shear stress modeling and suspension was collected in a Falcon tube and centrifuged
quantification, we also experimentally investigated the (1200 rpm for 5 min; CT6EL, Hitachi Koki, Tokyo,
impact of printing-associated shear stress and hydrostatic Japan). The supernatant was removed from the tube
pressure on cell viability and function in long-term cell and the remaining cell pellet was suspended with 10 ml
culture experiments. First, the viability of the HUVECs medium (EBM-2 Basal Medium and EGM-2 Endothelial
was measured after they were expelled from the printer. Growth SingleQuot Kit Supplement and Growth Factors,
Then, immune expression of cell specific proteins and Lonza, Basel, Switzerland). The cells were transferred to
the capillary-like network formation of printed HUVECs gelatin-coated cell culture flasks (CELLSTAR TC, sterile,
in a two-dimensional (2D) environment were compared Greiner Bio-One, Frickenhausen, Germany; Gelatin from
to results obtained from non-printed control samples. porcine skin, gel strength 300, Type A, Sigma-Aldrich,
Finally, post-printing tubular formation of HUVECs was St. Louis, USA) and incubated at 37°C and 5% v/v CO .
2
assessed in co-culture with hMSC in 3D agarose-collagen From then on, the medium was changed twice a week.
hydrogels after a period of 14 days. We hypothesized that 2.2. Preparation of hydrogel and PI-FDA solutions
bioprinting of pre-vascularized 3D constructs is feasible
without affecting the cell function and angiogenic The agarose (agarose low gelling temperature,
potential of HUVECs at a well-tuned hydrostatic pressure BioReagent for molecular biology, Sigma-Aldrich, St.
and resulting shear stress. Louis, USA) and alginate (alginic acid sodium salt from

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