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Mandt D, et al.

photoinitiator lithium-(2,4,6-trimethylbenzoyl)- methacrylization solution consisting of deionized
phenylphosphinate (Li-TPO-L), which was synthesized water (50% v/v), ethanol (48% v/v), glacial acetic acid
as previously published [14] . GelMOD-AEMA surfaces (0.3% v/v) and 3-(trimethoxysilyl)-propyl methacrylate
were prepared by applying drops of 50 µL warm gel (2% v/v) was prepared under continuous stirring. After a
solution on a pre-heated Teflon plate (40 °C). Previously contact time of 30 min, supernatant liquid was removed
methacrylated cover glasses were carefully placed on and coated glasses were washed with deionized-water
top of the droplets and gently pushed against the Teflon twice. After drying in a heating cabinet, slides were UV
plate, in order to guarantee a uniform coating. The sterilized.
Teflon plate with the coverslips was transferred into
the UV chamber and exposed to UV light for 10 min at 2.4 Fabrication of Microfluidic Devices
2
365 nm, which corresponds to 4 mW/cm . In the meantime, The complexity of the placental structure and the central
wells of a 12-well cell culture plate were coated with 1% requirement of two separately perfusable channels
agarose solution dissolved in PBS. Uncoated glasses and placed special demands on the chip geometry. Therefore,
glasses covered with a thin layer of GelMOD-AEMA in this study a custom-made microfluidic platform was
were gently em bedded, while the agarose was still used.
warm. Coating the wells with agarose prior to placing The chip had a rectangular footprint with dimensions
the glass slide ensures that cells exclusively stay on the of 76 × 26 mm and two adjacent chambers. Each
glass and not around or under the glass. Subsequently, chamber had four in-/outlets at its outermost points
un polymerized material was removed by washing with (Figure 1A). The molds of these chambers were
the respective cell culture media. Samples evaluating X-shaped with an intersection area of 1.4 by 1.0 mm
the effect of fibronectin-coated GelMOD-AEMA were creating an appropriate structure area. (Figure 1B). For
subsequently incubated with 50 µL/mL fibronectin for chip fabrication poly-(ethylene glycol)-dimethacrylate
4
30 min, at 37 °C. 20 × 10 HUVECs or BeWo B30 cells (PEGdma - Sigma Aldrich) with an average molecular
in 1 mL of the respective cell culture media were seeded weight of 700 was used. The photosensitive material
per well and allowed to attach overnight. solution, consisting of 40% v/v polymer solution,
Determination of metabolic activity was performed 0.6 mM Li-TPO-L photoinitiator and Dulbecco’s
24 and 48 hours post seeding using a resazurin-based phosphate buffered saline (DPBS - Sigma) was casted
staining solution. Cells were incubated for 90 min between a prepared glass chip. This chip was composed
®
at 37 °C with 100 µL Presto Blue (ThermoFisher) of three different layers: a 1 mm thick cover glass (Carl
solution which was prepared according to the manu- Roth) with four orifices, a 250 µm polydimethylsiloxane
facturer’s protocol. For absorbance readout, 100 µL (PDMS) spacer and a 170 µm thin bottom glass (ibidi).
super natant was transferred to a 96-well plate and ana- After chip frames were silanized with methacrylates, the
lyzed with a microplate reader (BioTek Instruments) injected PEGdma solution was irradiated for 50 seconds
using an excitation/emission filter of 570 nm and 600 nm, with an UV-LED (OmniCure) at 365 nm. After removal
res pectively. Furthermore, absolute DNA content of unpolymerized material, female luer lock connectors
was quantified 48 hours’ post seeding using the blue (ibidi) were glued on the orifices of the cover glass and
fluoro metric double-stranded DNA quantification kit the chip was sterilized by UV irradiation. All described
®
FluoReporter (Molecular Probes). fabrication steps were performed under sterile conditions
To evaluate changes in cell metabolism and DNA inside a lamina flow hood.
content of cells cultured in co-culture media, HUVECs 2.5 Preparation of the Placental Barrier Model
and BeWo B30 cells were seeded in 12-well plates
4
with an average concentration of 8 × 10 cells per Devices
well. To compare changes in growth, both cell types, 3D structures were produced within the sterile
HUVEC and BeWo B30, were either cultured in their microfluidic chip using an in-house built 2PP system
supplemented culture media EGM-2 and DMEM Ham (SI Figure 1) with a laser pulse length of 70 fs and a
F-12, res pectively, or cultivated in a 1:1 mixture of these repetition rate of 80 MHz at 800 nm (MaiTai, Spectra-
cell media. Changes in metabolic activity and DNA Physics). The system is similar to the one reported
content were evaluated as described above. previously . Due to the nonlinear behavior of 2PP, two-
[15]
2.3 Functionalization of Glass Surfaces with photon absorption triggers a local photopolymerization,
[15]
Methacrylate Groups with a feature size of less than 100 nm .
The laser beam was focused into the sample using a
For silanization 18 mm cover glasses (Carl Roth) 10x microscope objective, and scanned with a velocity
loaded in appropriate staining racks were pre-treated of 1 m/s and a laser power of 130 mW. Microfluidic
in a plasma cleaner (Harrick Plasma) for 10 min. The barrier structures were produced in the intersection of

International Journal of Bioprinting (2018)–Volume 4, Issue 2 3