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Patterning of tissue spheroids biofabricated from human fibroblasts on the surface of electrospun polyurethane matrix using…

is necessary to keep them close to each other in three- under voltage 17kV; the distance between needle end
dimensional space. Several different approaches have and collector was 20 cm; speed of polymer movement
been developed to enable controllable tissue spheroids was 1.3 mL/h; diameter of needle was 0.84 mm. Poly-
fusion, which include placing tissue sphe-roids inside urethane have been dissolved to concentration 17% in
3D printed synthetic scaffolds [11–13] , using bioprintable solvents containing 40% N,N-dimethylformamide (DMF)
hydrogel [14,15] and even metallic rods [16] . The search and 60% tetrahydrofuran (THF).
for the effective methods to keep tissue spheroids
close to each other during 3D bioprinting continues 2.2 Biomechanical Testing
and one of the possible perspective approaches is an Tensile tests were performed for electrospun polyure-
application of nanotechnology. It have been postulated thane material. Rectangular specimens (n = 5) were
in recent reviews that application of nanotechnology cut out using a template (two parallel blades). Dimen-
will enable biofabrication of complex human tissues sions of the trimmed specimens were: width — 5 mm;
and even organs [17,18] . Fabrication of nano-/microfib- length — 30 mm. The thickness of samples was mea-
rous synthetic scaffolds by electrospinning is one of sured using a cathetometer KLM-4 (Russia). The pre-
popular application of nanotechnology in tissue engi- cision of measurement was 0.001 mm. For tensile test
neering [19,20] . It has been demonstrated that tissue sph- Zwick-Roell BDO-FB0.5TS Test System (Germany)
eroids can attach, spread and fuse on synthetic elec- with load cell 50 N connected to PC was used. Sam-
trospun matrices [21,22] . ples were deformed with the speed of 5 mm/min until
Moreover, recently reported magnetic functionali- rupture. Maximal (failure) strain and maximal stress
zation of electrospun synthetic matrices with magnetic were estimated for each sample using TestExpert
nanoparticles [23] as well as biofabrication of tissue software Version 11.02 (Germany). The stiffness of
spheroids from cells labelled with magnetic nanopar- the material was assessed as the slope of the first li-
ticles [24–27] allow the development of magnetic forces- near range of the stress-strain curve, and was ex-
driven biofabrication and even 3D magnetic bioprint- pressed as a tangential modulus of elasticity.
ing based on principles of magnetic levitation [28–30] .
Thus, application of nanotechnology can enable devel- 2.3 Normal Human Dermal Fibroblast Cell Culture
opment of novel technology of magnetic 3D bioprinting.
We hypothesize that precise placing of tissue sphe- Normal human dermal fibroblasts (NHDF) were ob-
roids using 3D bioprinter on biocompatible electros- tained from Lonza (cat.# CC-2511). NHDF cells were
pun polyurethane matrix followed by their attachment grown in DMEM (Gibco, cat.# 12491-015) containing
and spreading will optimize biofabrication of tissue 10% FBS (Gibco, cat.# 16000-044) supplemented with
engineered constructions of desirable pattern and thi- antibiotic/antimycotic mix (Gibco, cat.# 15240-062),
ckness and allow the use of electrospun synthetic ma- 1 mM L-glutamine (Paneco, cat.# F032). The cells
trices as carrier for tissue spheroids. Thus, tissue en- were cultivated at 37ºC in humidified atmosphere with
gineered constructions formed by tissue spheroids 5% CO 2 and split at 85–95% confluence.
patterned, attached and spread on the surface of bio- 2.4 Biofabrication of Tissue Spheroids
compatible electrospun synthetic matrices could be
used as a novel technology platform in organ printing. The tissue spheroids were formed using the 3D petri
Reported spreading of patterned tissue spheroids could dishes (Microtissues, cat.# 12-81) according to manu-
be also used as an in vitro assay for testing biocompa- facturing protocol. Briefly, the 3D petri dishes were
tibility of various synthetic electrospun biomaterials. prepared from 2% agarose in PBS. NHDF monolayer
cells reached 95% confluence were rinsed by Versen
2. Materials and Methods (Paneco, cat.# R080), harvested from the culture fla-
sks by 0.25% trypsin — 0.53 mM EDTA (Gibco, cat.#
2.1 Electrospinning
25200-114) and then re-suspended in cell culture me-
Polyurethane was kindly provided by Dr Xuejun Wen dium. The concentrations of the NHDF cells were 6.8 ×
6
((EG-85A, Lubrizol, USA). Electrospinning of micro 10 per milliliter. 190 µL of cell suspension was see-
fibrous polyurethane matrix have been performed using ded into the 3D petri dishes. After 40 minutes addi-
commercial apparatus Professional Electrospinning Lab tional culture medium was added. The 3D petri dishes
Device (Yflow, Spain). Electrospinning was performed containing the tissue spheroids were incubated 4 days

46 International Journal of Bioprinting (2016)–Volume 2, Issue 1